Lipids for therapeutic agent delivery formulations

ABSTRACT

The description is directed to ionizable lipids useful for enhancing the delivery of therapeutic agents in liposomes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/657,480 filed Jun. 8, 2012, which is hereinincorporated by reference in its entirety.

TECHNICAL FIELD

The description is directed to ionizable lipids for enhancing thedelivery of therapeutic agents.

BACKGROUND

A number of techniques are available for delivering a therapeutic agent,for example, siRNA, nucleic acids, etc., into a cell. These techniquesinclude viral and non-viral transfection systems. Non-viral transfectionsystems can include, for example, polymers, lipids, liposomes, micelles,dendrimers, and nanomaterials. Polymers that have been studies for celltransfection include cationic polymers such as, for example,poly(L-lysine) (“PLL”), polyethyleneimine (“PEI”), chitosan, andpoly(2-dimethylamino)ethyl methacrylate (“pDMAEMA”).

The viral and non-viral transfection techniques have drawbacks, however.For example, viral systems can yield high transfection efficiency, butmay not be entirely safe. In addition, viral systems can be complicatedand/or expensive to prepare.

Non-viral transfection systems, for example, those employing cationicpolymers, have been reported to transfer plasmid DNA into cells.Cationic polymers, however, can be unstable and can be toxic to cells.

As such, there is a need for new compounds, compositions, and methodsfor using cationic composition to improve the delivery of therapeuticagents to cells, tissues, and organisms.

SUMMARY

The present description is directed to ionizable lipid compounds offormula I

wherein n and m are independently 1, 2, 3, or 4; R₁ and R₂ areindependently C₁₀₋₁₈ alkyl or C₁₂₋₁₈ alkenyl; X is —CH₂—, S, O, N, orabsent; L is C₁₋₄alkylene; —S—C₁₋₄ alkylene; —O—C₁₋₄ alkylene;—O—C(O)—C₁₋₄ alkylene; —S(O)₂—C₁₋₄ alkylene;

or a pharmaceutically acceptable salt form thereof. Compositions,pharmaceutical formulations, drug carriers, and methods of using thecompounds of formula I are also described.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts in vitro synergistic efficacy of ionizablelipid:ionizable lipid embodiments of the present description

FIG. 2 depicts in vitro synergistic efficacy of ionizablelipid:ionizable lipid embodiments of the present description

FIG. 3 depicts in vitro synergistic efficacy of ionizable lipid:cationiclipid embodiments of the present description

FIG. 4 depicts in vivo efficacy of ionizable lipid:cationic lipidembodiments of the present description

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present description is directed to ionizable lipid compounds, aswell as their uses in delivering therapeutic agents to cells, tissues,and organisms.

Within the scope of the description are ionizable lipid compounds offormula I:

wherein

n and m are independently 1, 2, 3, or 4;

R₁ and R₂ are independently C₁₀₋₁₈ alkyl or C₁₂₋₁₈ alkenyl;

X is —CH₂—, S, O, N, or absent;

L is C₁₋₄ alkylene; —S—C₁₋₄ alkylene; —O—C₁₋₄ alkylene; —O—C(O)—C₁₋₄alkylene; —S(O)₂—C₁₋₄ alkylene;

or a pharmaceutically acceptable salt form thereof.

Within the scope of the description, n and m can be the same ordifferent. In preferred embodiments, n and m are the same. Particularlypreferred are those embodiments wherein n and m are both 1 or n and mare both 2.

In some embodiments of the description, X is a bond. In otherembodiments, X is —CH₂—. In still others, X is S. Also preferred, areembodiments wherein X is O. Embodiments wherein X is N are also withinthe scope of the description.

In some embodiments of the description, L is C₁₋₄alkylene. In otherembodiments, L is —S—C₁₋₄ alkylene. In yet other embodiments, L is—O—C₁₋₄ alkylene. In still other embodiments, L is —O—C(O)—C₁₋₄alkylene. Alternatively, L is —S(O)₂—C₁₋₄ alkylene. Also within thescope of the description are embodiments wherein L is

In those embodiments wherein X is a bond, L is preferably C₁₋₄alkylene.Such examples of L include —CH₂—, —CH₂—CH₂—, —CH₂ CH₂CH₂—, and —CH₂CH₂CH₂CH₂—. In other embodiments wherein X is a bond, L is

for example,

In those embodiments wherein X is —CH₂—, L is preferably—S—C₁₋₄alkylene. Such examples of L include —S—CH₂—, —S—CH₂—CH₂—, —S—CH₂CH₂CH₂—, and —S—CH₂CH₂CH₂CH₂—.

In other embodiments wherein X is —CH₂—, L is preferably —S(O)₂—C₁₋₄alkylene. Such examples of L include —S(O)₂—CH₂, —S(O)₂—CH₂—CH₂,—S(O)₂—CH₂—CH₂—CH₂—, and S(O)₂—CH₂—CH₂—CH₂—CH₂—.

In yet other embodiments wherein X is —CH₂—, L is —O—C₁₋₄ alkylene. Suchexamples of L include —O—CH₂—, —O—CH₂—CH₂—, —O—CH₂ CH₂CH₂—, and —O—CH₂CH₂CH₂CH₂—.

In those embodiments of the description wherein X is S, L is preferablyC₁₋₄alkylene. Such examples of L include —CH₂—, —CH₂—CH₂—, —CH₂ CH₂CH₂—,and —CH₂CH₂CH₂CH₂—.

In those embodiments wherein X is O, L is preferably C₁₋₄alkylene. Suchexamples of L include —CH₂—, —CH₂—CH₂—, —CH₂ CH₂CH₂—, and —CH₂CH₂CH₂CH₂—.

Within the scope of the description, R₁ and R₂ can be the same ordifferent. Preferably, R₁ and R₂ are the same. Preferably, R₁ and R₂ areC₁₀₋₁₈ alkyl. Also preferred are embodiments wherein the C₁₀₋₁₈ alkyl isa straight-chain C₁₀₋₁₈ alkyl. More preferred are embodiments wherein R₁and R₂ are C₁₂₋₁₈ alkyl. Also preferred are embodiments wherein R₁ andR₂ are C₁₂₋₁₅ alkyl. In most preferred embodiments, R₁ and R₂ are bothC₁₋₃ alkyl.

In other embodiments of the description, R₁ and R₂ are C₁₂₋₁₈ alkenyl.Preferably, R₁ and R₂ are C₁₃₋₁₇ alkenyl. More preferably, R₁ and R₂ areeach oleyl:

In other embodiments of the description, R₁ and R₂ are C₁₂₋₁₈ alkenyl.Preferably, R₁ and R₂ are C₁₃₋₁₇ alkenyl. More preferably, R₁ and R₂ areeach linoleoyl.

Also within the scope of the description are compositions comprising acompound of formula I in a liposome, wherein the liposome comprises abilayer of lipid molecules. While the compound of formula I can compriseany mole percentage of the lipid molecules in such compositions, it ispreferred that the compound of formula I is about 5 to about 50 mol % ofthe lipid molecules of the compositions of the description.

Compositions of the description comprising a compound of formula I in aliposome can contain more than one compound of formula I. In preferredembodiments, such compositions of the description include two compoundsof formula I. In those embodiments, it is preferred that the molar ratioof the two compounds of formula I is about 10:30 to about 30:10.

Compositions of the description comprising a compound of formula I in aliposome may further comprise a cationic lipid. In such embodiments, thecationic lipid is about 5 to about 40 mol % of the lipid molecules ofthe composition. Also in these embodiments comprising a compound offormula I in a liposome with a cationic lipid, the molar ratio of thecompound of formula I to the cationic lipid is about 5:35 to about 35:5.More preferable, the ratio is about 10:30 to about 30:10.

Within the scope of the description, any compositions of the descriptionmay further comprise a liquid medium. Preferably, the liquid medium issuitable for injection into a living organism. In some embodiments, theliquid medium comprises an organic solvent. Alternatively, the liquidmedium used in certain embodiments of the description comprises waterand an organic solvent. In other embodiments of the description, theliquid medium may further comprise a non-aqueous medium.

Also within the scope of the description, any compositions of thedescription may further comprise at least one phospholipid.

In other embodiments of the description, any compositions of thedescription may further comprise at least one PEG-conjugated lipid.

Also within the scope of the description are stellate-cell-specific drugcarriers. These embodiments of the description include any of theaforementioned compositions, as well as a stellate cell specific amountof a targeting molecule consisting of a(retinoid)_(n)-linker-(retinoid)_(n), wherein n=0, 1, 2 or 3; andwherein the linker comprises a polyethylene glycol (PEG) or PEG-likemolecule.

In preferred embodiments, the drug carriers of the description willfurther comprising a siRNA molecule.

Also within the scope of the description are pharmaceuticalformulations. Pharmaceutical formulations within the scope of thedescription include any of the aforementioned drug carrier of thedescription and a pharmaceutically acceptable carrier or diluent. In ispreferred that in such formulations, the siRNA is encapsulated by theliposome of the compositions of the description.

Also within the scope of the description are methods of delivering adrug to a patient in need of treatment. These methods comprise providinga pharmaceutical formulation within the scope of the description andadministering the pharmaceutical formulation to the patient.

DEFINITIONS

The following terms are used throughout this specification.

As used herein, “cationic lipid” refers to a compound that includes atleast one lipid moiety and a positively charged quaternary nitrogenassociated with a counterion. “Lipids” are understood in the art to becomprises of a hydrophobic alkyl or alkenyl moiety and a carboxylic acidor ester moiety. Preferred cationic lipids for use in the presentdescription include:

As used herein, “ionizable lipid” refers to a compound of formula Iwithin the scope of the description. These compounds are capable offorming charged species when contacted with an appropriate counterionspecies, for example, a species that includes an ionizable hydrogenatom.

As used herein, “alkyl” refers to a straight or branched fully saturated(no double or triple bonds) hydrocarbon group, for example, a grouphaving the general formula —C_(n)H_(2n+1). The alkyl group may have 1 to50 carbon atoms (whenever it appears herein, a numerical range such as“1 to 50” refers to each integer in the given range; e.g., “1 to 50carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2carbon atoms, 3 carbon atoms, etc., up to and including 50 carbon atoms,although the present definition also covers the occurrence of the term“alkyl” where no numerical range is designated). The alkyl group mayalso be a medium size alkyl having 1 to 30 carbon atoms. The alkyl groupcould also be a lower alkyl having 1 to 5 carbon atoms. The alkyl groupof the compounds may be designated as “C₁₋₄ alkyl” or similardesignations. By way of example only, “C₁₋₄ alkyl” indicates that thereare one to four carbon atoms in the alkyl chain, i.e., the alkyl chainis selected from the group consisting of methyl, ethyl, propyl,isopropyl, n-butyl, isobutyl, sec-butyl, and t-butyl. Typical alkylgroups include, but are in no way limited to, methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl and the like.

As used herein, “alkylene” refers to an alkanediyl functional group, forexample, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂—, and the like.

As used herein, “alkenyl” refers to an alkyl group that contains in thestraight or branched hydrocarbon chain one or more double bonds. Analkenyl group may be unsubstituted or substituted. When substituted, thesubstituent(s) may be selected from the same groups disclosed above withregard to alkyl group substitution unless otherwise indicated. Oleyl isan example of such an alkenyl group.

As used herein, the term “pharmaceutical carrier” refers to a chemicalcompound that facilitates the incorporation of a compound into cells ortissues. For example dimethyl sulfoxide (DMSO) is a commonly utilizedcarrier as it facilitates the uptake of many organic compounds into thecells or tissues of an organism

As used herein, the term “diluent” refers to chemical compounds dilutedin water that will dissolve the formulation of interest (e.g., theformulation that can include a compound, a retinoid, a second lipid, astabilizing agent, and/or a therapeutic agent) as well as stabilize thebiologically active form of the formulation. Salts dissolved in bufferedsolutions are utilized as diluents in the art. One commonly usedbuffered solution is phosphate buffered saline because it mimics thesalt conditions of human blood. Since buffer salts can control the pH ofa solution at low concentrations, a buffered diluent rarely modifies thebiological activity of the formulation. As used herein, an “excipient”refers to an inert substance that is added to a formulation to provide,without limitation, bulk, consistency, stability, binding ability,lubrication, disintegrating ability, etc., to the composition. A“diluent” is a type of excipient.

“Organic solvents” used within the scope of the description are known inthe art, per se. and include, for example, C₁₋₄alkyl alcohols, dimethylsulfoxide (“DMSO”), and the like.

As used herein, “therapeutic agent” refers to a compound that, uponadministration to a mammal in a therapeutically effective amount,provides a therapeutic benefit to the mammal A therapeutic agent may bereferred to herein as a drug. Those skilled in the art will appreciatethat the term “therapeutic agent” is not limited to drugs that havereceived regulatory approval. A “therapeutic agent” can be operativelyassociated with a compound as described herein, a retinoid, and/or asecond lipid. For example, a second lipid as described herein can form aliposome, and the therapeutic agent can be operatively associated withthe liposome, e.g., as described herein.

As used herein, a “retinoid” is a member of the class of compoundsconsisting of four isoprenoid units joined in a head-to-tail manner, seeG. P. Moss, “Biochemical Nomenclature and Related Documents,” 2nd Ed.Portland Press, pp. 247-251 (1992). “Vitamin A” is the genericdescriptor for retinoids exhibiting qualitatively the biologicalactivity of retinol. As used herein, retinoid refers to natural andsynthetic retinoids including first generation, second generation, andthird generation retinoids. Examples of naturally occurring retinoidsinclude, but are not limited to, (1) 11-cis-retinal, (2) all-transretinol, (3) retinyl palmitate, (4) all-trans retinoic acid, and (5)13-cis-retinoic acids. Furthermore, the term “retinoid” encompassesretinols, retinals, retinoic acids, retinoids, and derivatives thereof.

As used herein, “retinoid conjugate” refers to a molecule that includesat least one retinoid moiety. In preferred embodiments of thedescription, the retinoid conjugate will be present at a concentrationof about 0.3 to about 30 weight percent, based on the total weight ofthe composition or formulation, which is equivalent to about 0.1 toabout 10 mol. %, which is equivalent to a molar ratio of about 0.1 toabout 10. Preferably, the retinoid conjugate is a retinoid-linker-lipidmolecule or a retinoid-linker-retinoid molecule.

An example of a retinoid conjugates include those compounds of formulaII:

wherein q, r, and s are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or10, and enantiomers and diastereomers thereof.

Preferred compounds of formula II include those wherein q, r, and s areeach independently 1, 2, 3, 4, 5, 6, or 7. More preferred are thosecompounds of formula II wherein q, r, and s are each independently 3, 4,or 5. Most preferred are those compounds of formula II wherein q is 3, ris 5, and s is 3. One example of a compound of formula II is

DiVA-PEG-DiVA includes stereocenters and all enantiomers anddiastereomers are considered to be within the scope of the description.

As used herein, “retinoid-linker-lipid molecule” refers to a moleculethat includes at least one retinoid moiety attached to at least onelipid moiety through at least one linker such as, for example, a PEGmoiety.

As used herein, “retinoid linker-retinoid molecule” refers to a moleculethat includes at least one retinoid moiety attached to at least oneother retinoid moiety (which may be the same or different) through atleast one linker such as, for example, a PEG moiety.

As used herein, the terms “lipid” and “lipophilic” are used herein intheir ordinary meanings as understood by those skilled in the art.Non-limiting examples of lipids and lipophilic groups include fattyacids, sterols, C₂-C₅₀ alkyl, C₂-C₅₀ heteroalkyl, C₂-C₅₀ alkenyl, C₂-C₅₀heteroalkenyl, C₂-C₅₀ aryl, C₂-C₅₀ heteroaryl, C₂-C₅₀ alkynyl, C₂-C₅₀heteroalkynyl, C₂-C₅₀ carboxyalkenyl, and C₂-C₅₀ carboxyheteroalkenyl. Afatty acid is a saturated or unsaturated long-chain monocarboxylic acidthat contains, for example, 12 to 24 carbon atoms A lipid ischaracterized as being essentially water insoluble, having a solubilityin water of less than about 0.01% (weight basis). As used herein, theterms “lipid moiety” and “lipophilic moiety” refers to a lipid orportion thereof that has become attached to another group. For example,a lipid group may become attached to another compound (e.g., a monomer)by a chemical reaction between a functional group (such as a carboxylicacid group) of the lipid and an appropriate functional group of amonomer.

As used herein, “stellate cell” refers to neurons with several dendritesradiating from the cell body. Examples of stellate cells are inhibitoryinterneurons.

As used herein, “siRNA” refers to small interfering RNA, also known inthe art as short interfering RNA or silencing RNA. siRNA is a class ofdouble stranded RNA molecules that have a variety of effects known inthe art, the most notable being the interference with the expression ofspecific genes and protein expression.

The term “liposome” is used herein in its ordinary meaning as understoodby those skilled in the art, and refers to a lipid bilayer structurethat contains lipids attached to polar, hydrophilic groups which form asubstantially closed structure in aqueous media. In some embodiments,the liposome can be operatively associated with one or more compounds,such as a therapeutic agent and a retinoid. A liposome may be comprisedof a single lipid bilayer (i.e., unilamellar) or it may comprised of twoor more lipid bilayers (i.e., multilamellar). While the interior of aliposome may consists of a variety of compounds, the exterior of theliposome is accessible to the aqueous formulation comprising theliposome. A liposome can be approximately spherical or ellipsoidal inshape.

In some embodiments, the siRNA will be encapsulated by the liposome sothat the siRNA is inaccessible to the aqueous medium. When encapsulatingsiRNA, the liposome will have a solid core; such liposomes encapsulatingsiRNA and having a solid core are termed “lipid nanoparticles” herein.In other embodiments, the siRNA will not be encapsulated by theliposome. In such embodiments, the siRNA can be complexed on the outersurface of the liposome by mixing preformed liposomes with RNA in anaqueous solution. In these embodiments, the siRNA is accessible to theaqueous medium. Liposomes having siRNA bound only on their outer surfaceare termed “lipoplexes” herein.

The formulations of the description can also include PEG-conjugatedlipids. PEG-conjugated lipids within the scope of the description areknown in the art per se. Suitable PEG-lipids include PEG-phospholipidsand PEG-ceramides such as, for example, PEG2000-DSPE, PEG2000-DPPE,PEG2000-DMPE, PEG2000-DOPE, PEG1000-DSPE, PEG1000-DPPE, PEG1000-DMPE,PEG1000-DOPE, PEG550-DSPE, PEG550-DPPE, PEG-550DMPE, PEG-1000DOPE,PEG-BML, PEG-Cholesterol. PEG2000-Ceramide, PEG1000-Ceramide,PEG750-Ceramide, PEG550-Ceramide.

The foregoing compositions of the description can include one or morephospholipids such as, for example,1,2-distearoyl-sn-glycero-3-phosphocholine (“DSPC”),dipalmitoylphosphatidylcholine (“DPPC”),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (“DPPE”), and1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (“DOPE”). Preferably, thehelper lipid is DOPE.

Also within the scope of the description are pharmaceutical formulationsthat include any of the aforementioned compositions in addition to apharmaceutically acceptable carrier or diluent. Pharmaceuticalformulations of the description will include at least one therapeuticagent. Preferably, the therapeutic agent is an siRNA. It is envisionedthat any siRNA molecule can be used within the scope of the description.For example, siRNA may include:

(SEQ. ID. NO. 1) Sense (5′->3′) GGACAGGCCUCUACAACUATT (SEQ. ID. NO. 2)Antisense (3′->5′) TTCCUGUCCGGAGAUGUUGAU and (SEQ. ID. NO. 3)Sense (5′->3′) GGACAGGCCUGUACAACUATT (SEQ. ID. NO. 4) Antisense (3′->5′)TTCCUGUCCGGACAUGUUGAU

In preferred formulations of the description including siRNA, the siRNAis encapsulated by the liposome. In other embodiments, the siRNA can beoutside of the liposome. In those embodiments, the siRNA can becomplexed to the outside of the liposome.

Also within the scope of the description are methods of delivering atherapeutic agent to a patient. These methods comprise providing apharmaceutical formulation including any of the foregoing compositionsand a pharmaceutically acceptable carrier or diluent; and administeringthe pharmaceutical formulation to the patient.

In another aspect, the present disclosure relates to a pharmaceuticalformulation comprising one or more physiologically acceptable surfaceactive agents, pharmaceutical carriers, diluents, excipients, andsuspension agents, or a combination thereof; and a formulation (e.g.,the formulation that can include a compound, a retinoid, a second lipid,a stabilizing agent, and/or a therapeutic agent) disclosed herein.Acceptable additional pharmaceutical carriers or diluents fortherapeutic use are well known in the pharmaceutical art, and aredescribed, for example, in Remington's Pharmaceutical Sciences, 18thEd., Mack Publishing Co., Easton, Pa. (1990), which is incorporatedherein by reference in its entirety. Preservatives, stabilizers, dyes,and the like may be provided in the pharmaceutical formulation. Forexample, sodium benzoate, ascorbic acid and esters of p-hydroxybenzoicacid may be added as preservatives. In addition, antioxidants andsuspending agents may be used. In various embodiments, alcohols, esters,sulfated aliphatic alcohols, and the like may be used as surface activeagents; sucrose, glucose, lactose, starch, crystallized cellulose,mannitol, light anhydrous silicate, magnesium aluminate, magnesiummetasilicate aluminate, synthetic aluminum silicate, calcium carbonate,sodium acid carbonate, calcium hydrogen phosphate, calcium carboxymethylcellulose, and the like may be used as excipients; coconut oil, oliveoil, sesame oil, peanut oil, soya may be used as suspension agents orlubricants; cellulose acetate phthalate as a derivative of acarbohydrate such as cellulose or sugar, or methylacetate-methacrylatecopolymer as a derivative of polyvinyl may be used as suspension agents;and plasticizers such as ester phthalates and the like may be used assuspension agents.

The pharmaceutical formulations described herein can be administered toa human patient per se, or in pharmaceutical formulations where they aremixed with other active ingredients, as in combination therapy, orsuitable pharmaceutical carriers or excipient(s). Techniques forformulation and administration of the compounds of the instantapplication may be found in “Remington's Pharmaceutical Sciences,” MackPublishing Co., Easton, Pa., 18th edition, 1990.

Suitable routes of administration may include, for example, parenteraldelivery, including intramuscular, subcutaneous, intravenous,intramedullary injections, as well as intrathecal, directintraventricular, intraperitoneal, intranasal, or intraocularinjections. The formulation (e.g., the formulation that can include acompound, a retinoid, a second lipid, a stabilizing agent, and/or atherapeutic agent) can also be administered in sustained or controlledrelease dosage forms, including depot injections, osmotic pumps, and thelike, for prolonged and/or timed, pulsed administration at apredetermined rate. Additionally, the route of administration may belocal or systemic.

The pharmaceutical formulations may be manufactured in a manner that isitself known, e.g., by means of conventional mixing, dissolving,granulating, dragee-making, levigating, emulsifying, encapsulating,entrapping or tableting processes.

Pharmaceutical formulations may be formulated in any conventional mannerusing one or more physiologically acceptable pharmaceutical carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Proper formulation is dependent upon the route of administration chosen.Any of the well-known techniques, pharmaceutical carriers, andexcipients may be used as suitable and as understood in the art; e.g.,in Remington's Pharmaceutical Sciences, above.

Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Suitableexcipients are, for example, water, saline, dextrose, mannitol, lactose,lecithin, albumin, sodium glutamate, cysteine hydrochloride, and thelike. In addition, if desired, the injectable pharmaceuticalformulations may contain minor amounts of nontoxic auxiliary substances,such as wetting agents, pH buffering agents, and the like.Physiologically compatible buffers include, but are not limited to,Hanks's solution, Ringer's solution, or physiological saline buffer. Ifdesired, absorption enhancing preparations may be utilized.

Pharmaceutical formulations for parenteral administration, e.g., bybolus injection or continuous infusion, include aqueous solutions of theactive formulation (e.g., the formulation that can include a compound, aretinoid, a second lipid, a stabilizing agent, and/or a therapeuticagent) in water-soluble form. Additionally, suspensions of the activecompounds may be prepared as appropriate oily injection suspensions.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents that increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multi-dose containers, with an added preservative. Theformulations may take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

In addition to the preparations described previously, the formulationsmay also be formulated as a depot preparation. Such long actingformulations may be administered by intramuscular injection. Thus, forexample, the formulations (e.g., the formulation that can include acompound, a retinoid, a second lipid, a stabilizing agent, and/or atherapeutic agent) may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

The compositions and formulations of the description may also beformulated for topical delivery and may be applied to the subject's skinusing any suitable process for application of topical delivery vehicle.For example, the formulation may be applied manually, using anapplicator, or by a process that involves both. Following application,the formulation may be worked into the subject's skin, e.g., by rubbing.Application may be performed multiple times daily or on a once-dailybasis. For example, the formulation may be applied to a subject's skinonce a day, twice a day, or multiple times a day, or may be applied onceevery two days, once every three days, or about once every week, onceevery two weeks, or once every several weeks.

Some embodiments herein are directed to a method of delivering atherapeutic agent to a cell. For example, some embodiments are directedto a method of delivering a therapeutic agent such as siRNA into a cell.Suitable cells for use according to the methods described herein includeprokaryotes, yeast, or higher eukaryotic cells, including plant andanimal cells (e.g., mammalian cells). In some embodiments, the cells canbe human fibrosarcoma cells (e.g., HT1080 cell line). In otherembodiments, the cells can be hepatic stellate cells (LX2 cell line). Inother embodiments, the cells can be cancer cells. In yet otherembodiments, the cells can be stem cells (pHSC cell line). Cell lineswhich are model systems for cancer may be used, including but notlimited to breast cancer (MCF-7, MDA-MB-438 cell lines), U87glioblastoma cell line, B16F0 cells (melanoma), HeLa cells (cervicalcancer), A549 cells (lung cancer), and rat tumor cell lines GH3 and 9L.In these embodiments, the formulations described herein can be used totransfect a cell. These embodiments may include contacting the cell witha formulation described herein that includes a therapeutic agent, tothereby deliver a therapeutic agent to the cell.

Disclosed herein are methods for treating a condition characterized byabnormal fibrosis, which may include administering a therapeuticallyeffective amount of a formulation described herein. Conditionscharacterized by abnormal fibrosis may include cancer and/or a fibroticdisease. Types of cancer that may be treated or ameliorated by aformulation described herein include, but are not limited to, lungcancer, pancreatic cancer, breast cancer, liver cancer, stomach cancer,and colon cancer. In an embodiment, the cancer that may be treated orameliorated is pancreatic cancer. In another embodiment, the cancer thatmay be treated or ameliorated is lung cancer. Types of fibrotic diseasethat may be treated or ameliorated by a formulation described hereininclude, but are not limited to, hepatic fibrosis, hepatic cirrhosis,pancreatitis, pancreatic fibrosis, cystic fibrosis, vocal cord scarring,vocal cord mucosal fibrosis, laryngeal fibrosis, pulmonary fibrosis,idiopathic pulmonary fibrosis, cystic fibrosis, myelofibrosis,retroperitoneal fibrosis, and nephrogenic systemic fibrosis. In anembodiment, the condition that may be treated or ameliorated is hepaticfibrosis.

The formulations or pharmaceutical compositions described herein may beadministered to the subject by any suitable means. Non-limiting examplesof methods of administration include, among others, (a) administrationvia injection, subcutaneously, intraperitoneally, intravenously,intramuscularly, intradermally, intraorbitally, intracapsularly,intraspinally, intrasternally, or the like, including infusion pumpdelivery; (b) administration locally such as by injection directly inthe renal or cardiac area, e.g., by depot implantation; as well asdeemed appropriate by those of skill in the art for bringing the activecompound into contact with living tissue.

Pharmaceutical compositions suitable for administration includeformulations (e.g., the formulation that can include a compound, aretinoid, a second lipid, a stabilizing agent, and/or a therapeuticagent) where the active ingredients are contained in an amount effectiveto achieve its intended purpose. The therapeutically effective amount ofthe compounds disclosed herein required as a dose will depend on theroute of administration, the type of animal, including human, beingtreated, and the physical characteristics of the specific animal underconsideration. The dose can be tailored to achieve a desired effect, butwill depend on such factors as weight, diet, concurrent medication andother factors which those skilled in the medical arts will recognize.More specifically, a therapeutically effective amount means an amount ofcompound effective to prevent, alleviate or ameliorate symptoms ofdisease or prolong the survival of the subject being treated.Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

As will be readily apparent to one skilled in the art, the useful invivo dosage to be administered and the particular mode of administrationwill vary depending upon the age, weight and mammalian species treated,the particular compounds employed, and the specific use for which thesecompounds are employed. The determination of effective dosage levels,that is the dosage levels necessary to achieve the desired result, canbe accomplished by one skilled in the art using routine pharmacologicalmethods. Typically, human clinical applications of products arecommenced at lower dosage levels, with dosage level being increaseduntil the desired effect is achieved. Alternatively, acceptable in vitrostudies can be used to establish useful doses and routes ofadministration of the compositions identified by the present methodsusing established pharmacological methods.

In non-human animal studies, applications of potential products arecommenced at higher dosage levels, with dosage being decreased until thedesired effect is no longer achieved or adverse side effects disappear.The dosage may range broadly, depending upon the desired effects and thetherapeutic indication. Typically, dosages may be about 10 microgram/kgto about 100 mg/kg body weight, preferably about 100 microgram/kg toabout 10 mg/kg body weight. Alternatively dosages may be based andcalculated upon the surface area of the patient, as understood by thoseof skill in the art.

The exact formulation, route of administration and dosage for thepharmaceutical compositions can be chosen by the individual physician inview of the patient's condition. (See e.g., Fingl et al. 1975, in “ThePharmacological Basis of Therapeutics”, which is hereby incorporatedherein by reference in its entirety, with particular reference to Ch. 1,p. 1). Typically, the dose range of the composition administered to thepatient can be from about 0.5 to about 1000 mg/kg of the patient's bodyweight. The dosage may be a single one or a series of two or more givenin the course of one or more days, as is needed by the patient. Ininstances where human dosages for compounds have been established for atleast some condition, the dosages will be about the same, or dosagesthat are about 0.1% to about 500%, more preferably about 25% to about250% of the established human dosage. Where no human dosage isestablished, as will be the case for newly-discovered pharmaceuticalcompositions, a suitable human dosage can be inferred from ED50 or ID50values, or other appropriate values derived from in vitro or in vivostudies, as qualified by toxicity studies and efficacy studies inanimals.

It should be noted that the attending physician would know how to andwhen to terminate, interrupt, or adjust administration due to toxicityor organ dysfunctions. Conversely, the attending physician would alsoknow to adjust treatment to higher levels if the clinical response werenot adequate (precluding toxicity). The magnitude of an administrateddose in the management of the disorder of interest will vary with theseverity of the condition to be treated and to the route ofadministration. The severity of the condition may, for example, beevaluated, in part, by standard prognostic evaluation methods. Further,the dose and perhaps dose frequency, will also vary according to theage, body weight, and response of the individual patient. A programcomparable to that discussed above may be used in veterinary medicine.

Although the exact dosage will be determined on a drug-by-drug basis, inmost cases, some generalizations regarding the dosage can be made. Thedaily dosage regimen for an adult human patient may be, for example, adose of about 0.1 mg to 2000 mg of each active ingredient, preferablyabout 1 mg to about 500 mg, e.g. 5 to 200 mg. In other embodiments, anintravenous, subcutaneous, or intramuscular dose of each activeingredient of about 0.01 mg to about 100 mg, preferably about 0.1 mg toabout 60 mg, e.g. about 1 to about 40 mg is used. In cases ofadministration of a pharmaceutically acceptable salt, dosages may becalculated as the free base. In some embodiments, the formulation isadministered 1 to 4 times per day. Alternatively the formulations may beadministered by continuous intravenous infusion, preferably at a dose ofeach active ingredient up to about 1000 mg per day. As will beunderstood by those of skill in the art, in certain situations it may benecessary to administer the formulations disclosed herein in amountsthat exceed, or even far exceed, the above-stated, preferred dosagerange in order to effectively and aggressively treat particularlyaggressive diseases or infections. In some embodiments, the formulationswill be administered for a period of continuous therapy, for example fora week or more, or for months or years.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety which are sufficient to maintain themodulating effects, or minimal effective concentration (MEC). The MECwill vary for each compound but can be estimated from in vitro data.Dosages necessary to achieve the MEC will depend on individualcharacteristics and route of administration. However, HPLC assays orbioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using MEC value. Compositionsshould be administered using a regimen which maintains plasma levelsabove the MEC for 10-90% of the time, preferably between 30-90% and mostpreferably between 50-90%.

In cases of local administration or selective uptake, the effectivelocal concentration of the drug may not be related to plasmaconcentration.

The amount of formulation administered may be dependent on the subjectbeing treated, on the subject's weight, the severity of the affliction,the manner of administration and the judgment of the prescribingphysician.

Formulations disclosed herein (e.g., the formulation that can include acompound, a retinoid, a second lipid, a stabilizing agent, and/or atherapeutic agent) can be evaluated for efficacy and toxicity usingknown methods. For example, the toxicology of a particular compound, orof a subset of the compounds, sharing certain chemical moieties, may beestablished by determining in vitro toxicity towards a cell line, suchas a mammalian, and preferably human, cell line. The results of suchstudies are often predictive of toxicity in animals, such as mammals, ormore specifically, humans. Alternatively, the toxicity of particularcompounds in an animal model, such as mice, rats, rabbits, or monkeys,may be determined using known methods. The efficacy of a particularcompound may be established using several recognized methods, such as invitro methods, animal models, or human clinical trials. Recognized invitro models exist for nearly every class of condition, including butnot limited to cancer, cardiovascular disease, and various immunedysfunction. Similarly, acceptable animal models may be used toestablish efficacy of chemicals to treat such conditions. When selectinga model to determine efficacy, the skilled artisan can be guided by thestate of the art to choose an appropriate model, dose, and route ofadministration, and regime. Of course, human clinical trials can also beused to determine the efficacy of a compound in humans.

The formulations may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration. The pack or dispensermay also be accompanied with a notice associated with the container inform prescribed by a governmental agency regulating the manufacture,use, or sale of pharmaceuticals, which notice is reflective of approvalby the agency of the form of the drug for human or veterinaryadministration. Such notice, for example, may be the labeling approvedby the U.S. Food and Drug Administration for prescription drugs, or theapproved product insert. Compositions comprising a compound formulatedin a compatible pharmaceutical carrier may also be prepared, placed inan appropriate container, and labeled for treatment of an indicatedcondition.

It is understood that, in any compound described herein having one ormore stereocenters, if an absolute stereochemistry is not expresslyindicated, then each center may independently be of R-configuration orS-configuration or a mixture thereof. Thus, the compounds providedherein may be enantiomerically pure or be stereoisomeric mixtures. Inaddition it is understood that, in any compound having one or moredouble bond(s) generating geometrical isomers that can be defined as Eor Z each double bond may independently be E or Z a mixture thereof.Likewise, all tautomeric forms are also intended to be included.

Preferred compounds of formula I within the scope of the description areset forth in Table 1. In vitro and in vivo data (see infra) is also setforth in Table 1.

TABLE 1 in vivo in vitro (rat (pHSC) DMNQ) Lipid Structure % KD % KDi-Pr-DC

53% @ 50 nM i-Pr- DODC

60% @ 50 nM i-DC

89% @ 200 nM i-Et-DC (Et104)

65% @ 50 nM i-Et- DODC

43% @ 50 nM i-Prop- DC

55% @ 50 nM i-Prop- DODC

55% @ 50 nM S104

68% @ 50 nM 52% @ 0.5 mpk S104- DO

78% @ 20 nM 65% @ 0.5 mpk C104

53% @ 20 nM 75% @ 0.5 mpk SO2- S104

75% @ 20 nM 18% @ 0.5 mpk TU104

76% @ 20 nM O104

55% @ 20 nM HEDC- M1

53% @ 20 nM C104- DO

32% @ 20 nM Pr104

54% @ 20 nM Pr104- DO

27% @ 20 nM T104

58% @ 20 nM TU104- DO

81% @ 20 nM 40% @ 0.25 mpk CB104

42% @ 20 nM CA104

INT-4

70% @ 50 nM S104- DMO

40% @ 10 nM Pro-DC

S104- DLin

TU104- DLin

The description can be further exemplified by reference to the followingexamples. These examples are illustrative, only, and are not intended tolimit the description.

EXPERIMENTAL SECTION Preparation of((2-(dimethylamino)acetyl)azanediyl)bis(propane-3,1-diyl)ditetradecanoate(i-Pr-DC)

Step 1: Preparation of Intermediate 1: 3,3′-azanediylbis(propan-1-ol)

A mixture of 3-amino-1-propanol (14.5 mL, 19.0 mmol), 1-chloro-3-hydroxypropane (8.00 mL, 95.6 mmol) and H₂O (˜50 mL) was refluxed over 24hours. Potassium hydroxide (5.40 g) was then added. After dissolution,the whole of the water was evaporated to leave viscous oil and largequantities of potassium chloride. These were filtered and washed withdry acetone and dichloromethane. The organic phase was dried overNa₂SO₄, filtered and evaporated to leave an oil. Purification by silicagel chromatography eluating with a DCM/MeOH gradient yielded3,3′-azanediylbis(propan-1-ol) (12.5 g).

Step 2: Preparation of Intermediate 2: tert-butylbis(3-hydroxypropyl)carbamate

3,3′-azanediylbis(propan-1-ol) (12.5 g, 95.4 mmol) was diluted in DCM(25 mL). A solution of di-tert-butyl dicarbonate (26.0 g, 119 mmol) inDCM (25 mL) was slowly added while stirring under a blanket of argongas. Reaction was allowed to stir overnight. The reaction mixture wasconcentrated. Purification by silica gel chromatography eluating with aDCM/MeOH gradient yielded tert-butyl bis(3-hydroxypropyl)carbamate.

Step 3: Preparation of Intermediate 3:((tert-butoxycarbonyl)azanediyl)bis(propane-3,1-diyl)ditetradecanoate

tert-butyl bis(3-hydroxypropyl)carbamate (4.00 g, 17.3 mmol), Et3N (4.8mL, 34.6 mmol) and DMAP (529 mg, 4.33 mmol) were dissolved in chloroform(50 mL). While being stirred in an ice-bath, a solution of myristoylchloride was added over 15 min. The addition was carried out in such away that the temperature of the reaction did not exceed 30° C. Thereaction was allowed to stir at room temperature overnight. Next day,MeOH (50 mL) and 0.9% saline solution (50 mL) was added to quench thereaction. The organic layer was separated and washed with 1M NaHCO3.Solvent was dried with Na2SO4, filtered and concentrated in vacuo toyield((tert-butoxycarbonyl)azanediyl)bis(propane-3,1-diyl)ditetradecanoate asan oil that was carried forward without further purification.

Step 4: Preparation of Intermediate 4:azanediylbis(propane-3,1-diyl)ditetradecanoate TFA salt

((tert-butoxycarbonyl)azanediyl)bis(propane-3,1-diyl)ditetradecanoate(11.3 g, 17.3 mmol) was dissolved in TFA/CHCl3 (1:1, 20 mL) and themixture was allowed to stir at room temperature for 15 minutes. Materialwas then concentrated in vacuo. This was repeated a second time.Material was then dissolved in DCM and washed with H₂O, dried withNa₂SO₄, concentrated in vacuo and dried fully overnight. The reactionmixture was concentrated. Purification by silica gel chromatographyeluating with a DCM/MeOH gradient yieldedazanediylbis(propane-3,1-diyl)ditetradecanoate TFA salt (7.5 g).

Step 5: Preparation of i-Pr-DC:((2-(dimethylamino)acetyl)azanediyl)bis(propane-3,1-diyl)ditetradecanoate

Azanediylbis(propane-3,1-diyl)ditetradecanoate TFA salt (750 mg, 1.35mmol) was diluted with DCM (5 mL) and added to a pre-activated mixtureof N,N-dimethylglycine (154 mg, 1.49 mmol), HATU (616 mg, 1.62 mmol) andDIEA (495 uL, 2.84 mmol) in DCM (5 mL). Flask was flushed with argon andallowed to stir at room temperature overnight. The reaction mixture wasconcentrated. Purification by silica gel chromatography eluating with aDCM/MeOH gradient yielded((2-(dimethylamino)acetyl)azanediyl)bis(propane-3,1-diyl)ditetradecanoate(465 mg). QTOF MS ESI+: m/z 639.6 (M+H).

Preparation of(Z)-((2-(dimethylamino)acetyl)azanediyl)bis(propane-3,1-diyl)dioleate(i-Pr-DODC)

Step 1: Preparation of Intermediate 1: 3,3′-azanediylbis(propan-1-ol

A mixture of 3-amino-1-propanol (14.5 mL, 19.0 mmol), 1-chloro-3-hydroxypropane (8.00 mL, 95.6 mmol) and water (50 mL) was refluxed over 24hours. Potassium hydroxide (5.40 g) was then added. After dissolution,the whole of the water was evaporated to leave a viscous oil and largequantities of potassium chloride. These were filtered and washed withdry acetone and dichloromethane. The organic phase was dried overNa₂SO₄, filtered and evaporated to leave an oil. Purification by silicagel chromatography eluating with a DCM/MeOH gradient yielded3,3′-azanediylbis(propan-1-ol) (12.5 g).

Step 2: Preparation of Intermediate 2: tert-butylbis(3-hydroxypropyl)carbamate

3,3′-azanediylbis(propan-1-ol) (12.5 g, 95.4 mmol) was diluted in DCM(25 mL). A solution of di-tert-butyl dicarbonate (26.0 g, 119 mmol) inDCM (25 mL) was slowly added while stirring under a blanket of Ar gas.Reaction was allowed to stir overnight. The reaction mixture wasconcentrated. Purification by silica gel chromatography eluating with aDCM/MeOH gradient yielded tert-butyl bis(3-hydroxypropyl)carbamate.

Step 3: Preparation of Intermediate 3:(Z)-((tert-butoxycarbonyl)azanediyl)bis(propane-3,1-diyl)dioleate

tert-butyl bis(3-hydroxypropyl)carbamate, triethylamine and DMAP weredissolved in chloroform. While being stirred in an ice-bath, a solutionof oleyl chloride was added over 15 minutes. The addition was carriedout in such a way that the temperature of the reaction did not exceed30° C. The reaction was allowed to stir at room temperature overnight.Next day, MeOH (50 mL) and 0.9% saline solution (50 mL) was added toquench the reaction. The organic layer was separated and washed with 1MNaHCO₃. Solvent was dried with Na₂SO₄, filtered and concentrated invacuo to yield(Z)-((tert-butoxycarbonyl)azanediyl)bis(propane-3,1-diyl)dioleate as anoil that was carried forward without further purification.

Step 4: Preparation of Intermediate 4:(Z)-azanediylbis(propane-3,1-diyl)dioleate TFA salt

(Z)-((tert-butoxycarbonyl)azanediyl)bis(propane-3,1-diyl)dioleate (13.2g, 17.3 mmol) was dissolved in TFA/CHCl₃ (1:1, 20 mL) and the mixturewas allowed to stir at room temperature for 15 minutes. Material wasthen concentrated in vacuo. This was repeated a second time. Materialwas then dissolved in DCM and washed with H₂O, dried with Na₂SO₄ andconcentrated in vacuo. Purification by silica gel chromatographyeluating with a DCM/MeOH gradient yielded(Z)-azanediylbis(propane-3,1-diyl)dioleate TFA salt.

Step 5: Preparation of i-Pr-DODC:(Z)-((2-(dimethylamino)acetyl)azanediyl(bis(propane-3,1-diyl)dioleate

(Z)-azanediylbis(propane-3,1-diyl)dioleate TFA salt (750 mg, 1.13 mmol)was diluted with DCM (5 mL) and added to a pre-activated mixture ofN,N-dimethylglycine (128 mg, 1.24 mmol), HATU (517 mg, 1.36 mmol) andDIEA (413 uL, 2.37 mmol) in DCM (5 mL). Flask was flushed with argon andallowed to stir at room temperature overnight. The reaction mixture wasconcentrated. Purification by silica gel chromatography eluating with aDCM/MeOH gradient yielded(Z)-((2-(dimethylamino)acetyl)azanediyl)bis(propane-3,1-diyl)dioleate(450 mg). QTOF MS ESI+: m/z 747.7 (M+H).

Preparation of((2-(dimethylamino)acetyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate,(i-DC)

Step 1: Preparation of Intermediate 1:((tert-butoxycarbonyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate

N-Boc diethanolamine (MW 205.25; 8.4 g, 0.041 mole), triethylamine (MW101.19; 11.5 ml, 0.083 mol) and 4-(dimethylamino)pyridine (MW 122.17;1.3 g, 0.011 mole) were dissolved in chloroform (170 mL). While beingstirred in an ice/water bath, a solution of myristoyl chloride (MW246.82; 22 mL, 80.9 mmol) in 100 mL of chloroform was added dropwise.The reaction mixture was then taken out of the cold bath, and thestirring was continued at room temperature for 2 h. A mixture of 200 mLof methanol and 200 ml of 0.9% saline was added to quench the reaction.The stirring was stopped and the organic layer was isolated. The solventwas removed by rotary evaporation to afford((tert-butoxycarbonyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate asa colorless oil (25.7 g) that was carried forward without furtherpurification.

Step 2: Preparation of Intermediate 2:azanediylbis(ethane-2,1-diyl)ditetradecanoate TFA salt

To a solution of((tert-butoxycarbonyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate(33.0 g, 0.053 mole) in 100 ml of chloroform was added trifluoroaceticacid (150 mL, 2.02 mol). The reaction mixture was stirred at roomtemperature overnight. After the solvent was removed by rotaryevaporation, the resultant soft solid was recrystallized from 80 ml ofmethanol to yield azanediylbis(ethane-2,1-diyl)ditetradecanoate TFA salt(16.6 g) as a white solid.

Step 3: Preparation of i-DC:((2-(dimethylamino)acetyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate

Azanediylbis(ethane-2,1-diyl)ditetradecanoate TFA salt (10 g, 16 mmol)was diluted with dimethylglycine (42.5 g, 25 mmol), DCC (4.7 g, 23 mmol)and DIEA (6.33 mL, 40 mmol) in Pyridine (20 mL). The round-bottomedflask was flushed with argon gas and the reaction mixture was heated at55° C. overnight. Next day, the reaction mixture was concentrated. Afterpurification by silica gel chromatography eluating with a DCM/MeOH, thepooled fractions were concentrated to yield((2-(dimethylamino)acetyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate.

Preparation of((3-(dimethylamino)propanoyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate(i-Et-DC, also referred to herein as Et104)

Preparation of i-Et-DC:((3-(dimethylamino)propanoyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate

Synthesis of azanediylbis(ethane-2,1-diyl)ditetradecanoate TFA saltpreviously described. Azanediylbis(ethane-2,1-diyl)ditetradecanoate TFAsalt (1.50 g, 2.85 mmol) was diluted with DCM (10 mL) and added to apre-activated mixture of 3-(dimethylamino)propionic acid HCl salt (482mg, 3.14 mmol), HATU (1.30 g, 3.42 mmol) and DIEA (1.04 mL, 5.98 mmol)in DCM (10 mL). The round-bottomed flask was flushed with argon gas andthe reaction mixture was allowed to stir at room temperature overnight.The reaction mixture was concentrated. After purification by silica gelchromatography eluating with a DCM/MeOH, the pooled fractions wereconcentrated and stirred in DCM (20 mL) and 10% K₂CO₃ (20 mL) at 0-5° C.for 30 min. The organic layer was isolated and the aqueous layer furtherextracted with DCM (2×10 mL). The combined organics were stirred withMgSO₄ for 30 min at 0-5° C., filtered, washed with DCM, and concentratedto yield((3-(dimethylamino)propanoyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate(1.01 g). QTOF MS ESI+: m/z 625.6 (M+H).

Preparation of(Z)-((3-(dimethylamino)propanoyl)azanediyl)bis(ethane-2,1-diyl)dioleatei-Et-DODC

Step 1: Preparation of Intermediate 1:(Z)-((tert-butoxycarbonyl)azanediyl)bis(ethane-2,1-diyl)dioleate

N-Boc diethanolamine (17.8 g, 0.087 mole), triethylamine (24.4 mL, 0.176mole) and 4-(dimethylamino)pyridine (2.76 g, 0.023 mole) were dissolvedin 350 ml of chloroform. While being stirred, a solution of oleylchloride (61.6 g, 0.174 mole) in 100 ml of chloroform was added over 10min (Alternatively, the chloroform solution of N-Boc diethanolamine wasimmersed in an ice/water bath while oleyl chloride was added). Theaddition was carried out in such a way that the temperature of thereaction mixture does not exceed 50° C. The reaction mixture was stirredat room temperature for 2 hrs. A mixture of 200 ml of methanol and 200ml of 0.9% saline was added to quench the reaction. The organic layerwas separated and washed with 2×100 ml of dilute aqueous sodiumbicarbonate. The solvent was removed by rotary evaporation to afford(Z)-((tert-butoxycarbonyl)azanediyl)bis(ethane-2,1-diyl)dioleate as apale yellow oil (59.5 g). This material was used for the next stepwithout further purification. 1H NMR (400 MHz, CDCl3) 0.87 (t, 6H, CH3),1.20-1.40 (m, 40H, CH2), 1.45 (s, 9H, tBu CH3), 1.59 (m, 4H,CH2CH2C(═O)), 2.00 (m, 8H, CH2CH═CH), 2.33 (t, 4H, CH2C(═O)), 3.48 (m,4H, NCH2CH2O), 4.18 (m, 4H, NCH2CH2O), 5.33 (m, 4H, CH═CH).

Step 2: Preparation of Intermediate 2:(Z)-azanediylbis(ethane-2,1-diyl)dioleate TFA salt

(Z)-((tert-butoxycarbonyl)azanediyl)bis(ethane-2,1-diyl)dioleate (59.5g, 0.081 mole) was treated twice with 100 ml trifluoroacetic acid (100mL, 1.35 mol) and 100 ml of chloroform. Each consisted of stirring atroom temperature for 10 min, and the solvent was removed by rotaryevaporation at the end of each treatment. The residue was dissolved in200 ml of methylene chloride and the mixture had been washed with 100 mlof water twice. The residue was purified by the silica gelchromatography using a mixture of methanol and methylene chloride aseluent to yield (Z)-azanediylbis(ethane-2,1-diyl)dioleate TFA salt (44.0g). 1H NMR (400 MHz, CDCl₃) 0.87 (t, 6H, CH₃), 1.20-1.40 (m, 40H, CH₂),1.59 (m, 4H, CH₂CH₂C(═O)), 2.00 (m, 8H, CH₂CH═CH), 2.33 (t, 4H,CH₂C(═O)), 3.31 (m, 4H, NCH₂CH₂O), 4.38 (m, 4H, NCH₂CH₂O), 5.33 (m, 4H,CH═CH).

Step 3: Preparation of i-Et-DODC:(Z)-((3-(dimethylamino)propanoyl)azanediyl)bis(ethane-2,1-diyl)dioleate

(Z)-azanediylbis(ethane-2,1-diyl)dioleate TFA salt (1.50 g, 2.37 mmol)was diluted with DCM (10 mL) and added to a pre-activated mixture of3-(dimethylamino)propionic acid HCl salt (383 mg, 2.49 mmol), HATU (1.03g, 2.72 mmol) and DIEA (831 uL, 4.77 mmol) in DCM (10 mL). Theround-bottomed flask was flushed with argon gas and the reaction mixturewas allowed to stir at room temperature overnight. The reaction mixturewas concentrated. After purification by silica gel chromatographyeluating with a DCM/MeOH, the pooled fractions were concentrated andstirred in DCM (20 mL) and 10% K₂CO₃ (20 mL) at 0-5° C. for 30 min. Theorganic layer was isolated and the aqueous layer further extracted withDCM (2×10 mL). The combined organics were stirred with MgSO₄ for 30 minat 0-5° C., filtered, washed with DCM, and concentrated to yield(Z)-((3-(dimethylamino)propanoyl)azanediyl)bis(ethane-2,1-diyl)dioleate.QTOF MS ESI+: m/z 733.6 (M+H).

Preparation of((4-(dimethylamino)butanoyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate[i-Prop-DC (also referred to herein as Pr104)]

Synthesis of azanediylbis(ethane-2,1-diyl)ditetradecanoate TFA saltpreviously described. Azanediylbis(ethane-2,1-diyl)ditetradecanoate TFAsalt (1.00 g, 1.90 mmol) was diluted with DCM (5 mL) and added to apre-activated mixture of 4-(Dimethylamino) butyric acid HCl salt (382mg, 2.28 mmol), HATU (867 mg, 2.28 mmol) and DIEA (728 uL, 4.18 mmol) inDCM (5 mL). The round-bottomed flask was flushed with argon gas and thereaction mixture was allowed to stir at room temperature overnight. Thereaction mixture was concentrated. After purification by silica gelchromatography eluating with a DCM/MeOH, the pooled fractions wereconcentrated and stirred in DCM (20 mL) and 10% K₂CO₃ (20 mL) at 0-5° C.for 30 min. The organic layer was isolated and the aqueous layer furtherextracted with DCM (2×10 mL). The combined organics were stirred withMgSO₄ for 30 min at O-SoC, filtered, washed with DCM, and concentratedto yield((4-(dimethylamino)butanoyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate.LCMS ESI+: m/z 639.6 (M+H).

Preparation of(Z)-((4-(dimethylamino)butanoyl)azanediyl)bis(ethane-2,1-diyl)dioleate[i-Prop-DODC (also referred to herein as Pr104-DO)]

Synthesis of (Z)-azanediylbis(ethane-2,1-diyl)dioleate TFA saltpreviously described. (Z)-azanediylbis(ethane-2,1-diyl)dioleate TFA salt(1.00 g, 1.58 mmol) was diluted with DCM (5 mL) and added to apre-activated mixture of 4-(Dimethylamino) butyric acid HCl salt (317mg, 1.89 mmol), HATU (719 mg, 1.89 mmol) and DIEA (606 uL, 3.48 mmol) inDCM (5 mL). The round-bottomed flask was flushed with argon gas and thereaction mixture was allowed to stir at room temperature overnight. Thereaction mixture was concentrated. After purification by silica gelchromatography eluating with a DCM/MeOH, the pooled fractions wereconcentrated and stirred in DCM (20 mL) and 10% K₂CO₃ (20 mL) at 0-5° C.for 30 min. The organic layer was isolated and the aqueous layer furtherextracted with DCM (2×10 mL). The combined organics were stirred withMgSO₄ for 30 min at 0-5° C., filtered, washed with DCM, and concentratedto yield(Z)-((4-(dimethylamino)butanoyl)azanediyl)bis(ethane-2,1-diyl)dioleate.LCMS ESI+: m/z 747.7 (M+H).

Preparation of(Z)-((2-((2-(dimethylamino)ethyl)thio)acetyl)azanediyl)bis(ethane-2,1-diyl)dioleate(S104-DO)

Synthesis of (Z)-azanediylbis(ethane-2,1-diyl)dioleate TFA saltpreviously described. (Z)-azanediylbis(ethane-2,1-diyl)dioleate TFA salt(4.06 g, 6.41 mmol) was stirred in DCM (60 mL) with 10% K₂CO₃ (30 mL) at0-5° C. After 30 min, the organic phase is separated and the aqueousphase was further extracted with DCM (30 mL). The combined organicphases are stirred with MgSO₄ for a period of 30 min at 0-5° C.,filtered and washed with DCM (−30 mL). To the combined filtrates wereadded 2-((2-(dimethylamino)ethyl)thio)acetic acid (1.26 g, 7.70 mmol),EDC HCl salt (1.84 g, 9.62 mmol), DMAP (78.3 mg, 0.64 mmol) and the thinsuspension was stirred overnight at room temperature. Next day, H₂O (60mL) and MeOH (30 mL) are added and after stirring for 10 min, the clearorganic phase was isolated. The turbid aqueous phase was extracted withDCM. The combined organic extracts are concentrated. Crude material wasfiltered through a plug of silica and taken up in DCM (40 mL) and PBS(pH=11, 50 mL) was added. The mixture was stirred at room temperaturefor ˜10 min. Afterwards, the organic phase was separated and the aqueousphase was extracted again with DCM (15 mL). The combined organics aredried (MgSO₄) for 30 min, filtered, washed with DCM, and concentrated toyield(Z)-((2-((2-(dimethylamino)ethyl)thio)acetyl)azanediyl)bis(ethane-2,1-diyl)dioleate(3.44 g). LCMS ESI+: m/z 780.2 (M+H).

Preparation((5-(dimethylamino)pentanoyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate(C104)

Synthesis of azanediylbis(ethane-2,1-diyl)ditetradecanoate TFA saltpreviously described. Azanediylbis(ethane-2,1-diyl)ditetradecanoate TFAsalt (730 mg, 1.14 mmol) was stirred in DCM (20 mL) with 10% K₂CO₃ (10mL) at 0-5° C. After 30 min, the organic phase was separated and theaqueous phase was further extracted with DCM (10 mL). The combinedorganic phases are stirred with MgSO₄ for a period of 30 minutes at 0-5°C., filtered and washed with DCM (10 mL). To the combined filtrates areadded 5-(dimethylamino)pentanoic acid (248 mg, 1.37 mmol), EDC HCl salt(328 mg, 1.71 mmol), DMAP (14 mg, 0.114 mmol) and the thin suspensionwas stirred overnight at room temperature, after which the solutionbecomes clear. Next day, H₂O (20 mL) and MeOH (10 mL) are added andafter stirring for 10 min, the clear organic phase was isolated. Theturbid aqueous phase was extracted with DCM. The combined organicextracts are concentrated. After purification by silica gelchromatography by eluating with 100% ethyl acetate followed by 10%MeOH/DCM, the purified residue was taken up in DCM (25 mL) and PBS(pH=11, 25 mL). The mixture was stirred at room temperature for 15 min.Afterwards, the organic phase was separated out and the aqueous phasewas extracted again with DCM (15 mL). The combined organics are dried(MgSO₄) for 30 min, filtered, washed with DCM, and concentrated to yield((5-(dimethylamino)pentanoyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate(405 mg). LCMS ESI+: m/z 654.1 (M+H).

Preparation of((2-((2-(dimethylamino)ethyl)sulfonyl)acetyl)azanediyl)bis(ethane-2,1-diyl)ditetra-decanoate(SO2-S104)

Synthesis of((2-((2-(dimethylamino)ethyl)thio)acetyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate,aka S104, was described. To((2-((2-(dimethylamino)ethyl)thio)acetyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoatein a round-bottom flask, flushed with argon was added DCM (10 mL). Thesolution was cooled by an ice-bath. To this, was added mCPBA (a solutionin DCM) slowly over 5 min. The ice bath was removed after the additionand the reaction was allowed to stir overnight at room temperature.After 3.5 hours, 2M DMA/THF (4.55 mL) was slowly added and the reactionmixture was allowed to stir overnight. The reaction mixture was thendiluted with DCM to 75 mL. Washed with H₂O (2×50 mL) and 10% K₂CO₃ (50mL). Back extracted all aqueous washes with DCM (40 mL). The combinedorganics were dried (MgSO₄), filtered, and concentrated to yield acolorless oil. The reaction mixture was concentrated. Purification bysilica gel chromatography eluating with a ethyl acetate/MeOH gradientyielded((2-((2-(dimethylamino)ethyl)sulfonyl)acetyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate(540 mg). LCMS ESI+: m/z 704.0 (M+H).

Preparation of((((2-(dimethylamino)ethyl)thio)carbonyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate(TU104)

Step 1: Preparation of Intermediate 1:azanediylbis(ethane-2,1-diyl)ditetradecanoate

Synthesis of azanediylbis(ethane-2,1-diyl)ditetradecanoate TFA saltpreviously described. Azanediylbis(ethane-2,1-diyl)ditetradecanoate TFAsalt was dissolved in DCM (50 mL) and PBS (pH=11, 50 mL) was added. Themixture was stirred at room temperature for 15 min. Afterwards, theorganic phase separated and the aqueous phase extracted again with DCM(25 mL). The combined organics were dried (MgSO₄) for 30 min, filtered,washed with DCM, and concentrated to yieldazanediylbis(ethane-2,1-diyl)ditetradecanoate as the free base.

Step 2: Preparation of TU104:((((2-(dimethylamino)ethyl)thio)carbonyl)azanediyl)bis(ethane-2,1-diyl)ditetra-decanoate

Trichloromethyl chloroformate (aka diphosgene) (257 uL, 2.13 mmol) wasadded to a solution of 2-(dimethylamino)ethanethiol HCl salt (302 mg,2.13 mmol) in dry DCM (20 mL) and stirred under a blanket of argon atroom temperature for 4 h. Afterwards, DCM and excess diphosgene wereremoved in vacuo. Azanediylbis(ethane-2,1-diyl)ditetradecanoate freebase (1068 mg, 2.03 mmol), DCM (20 mL) and triethylamine (580 uL, 4.16mmol) were then added. After 16 h at room temperature the reactionmixture was diluted with DCM and washed with 1M HCl (75 mL), H2O (75 mL)and PBS (pH=11, 75 mL), dried (MgSO₄), filtered, and concentrated.Purification by silica gel chromatography eluating with ethyl acetatefollowed by a DCM/MeOH gradient yielded((((2-(dimethylamino)ethyl)thio)carbonyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate(120 mg). LCMS ESI+: m/z 657.5 (M+H).

Preparation of((2-(2-(dimethylamino)ethoxy)acetyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate(O104)

Step 1: Preparation of Intermediate 1:((2-bromoacetyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate

Synthesis of azanediylbis(ethane-2,1-diyl)ditetradecanoate TFA saltpreviously described. Azanediylbis(ethane-2,1-diyl)ditetradecanoate TFAsalt (1500 mg, 2.34 mmol) was dissolved in DCM (20 mL) and placed in anice-bath. Bromoacetyl bromide (214 uL, 2.46 mmol) was added followed bytriethylamine (685 uL, 4.91 mmol). The ice-bath was removed and thereaction was allowed to stir overnight at room temperature under ablanket of inert gas. Next day, diluted with DCM to 100 mL. Washed with1M HCl (75 mL), H₂O (75 ml), saturated NaHCO₃ solution (75 mL) andsaturated brine solution (75 mL). Back extracted all aqueous washes withDCM (25 mL). Dried organics with MgSO₄, filtered and concentrated invacuo. Purified by silica gel chromatography and eluating with 100%ethyl acetate. Pooled and concentrated fractions to yield((2-bromoacetyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate (1220mg).

Step 2: Preparation of O104:((2-(2-(dimethylamino)ethoxy)acetyl)azanediyl)bis(ethane-2,1-diyl)ditetradeca-noate

To a round-bottom flask equipped with stir bar, was added((2-bromoacetyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate (1.22 g,1.87 mmol), N,N-dimethylethanolamine (197 uL, 1.96 mmol), potassiumiodide (6.2 mg, 0.0374 mmol) and dry THF (25 mL). The resulting solutionwas cooled to −40° C. DBU (588 uL, 3.93 mmol) was added dropwise over 5min and the reaction was warmed to 0° C. for 2 hours. The reactionmixture was concentrated. The residue was taken up with DCM, 1M HCl (12mL) was added, and biphasic mixture stirred for 15 min. Then, basifiedusing PBS (pH=11). The organic layer was isolated, dried (MgSO₄),filtered and concentrated. Purification by silica gel chromatographyeluating with 100% ethyl acetate followed by a DCM/MeOH gradient yielded((2-(2-(dimethylamino)ethoxy)acetyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate(53 mg). LCMS ESI+: m/z 655.6 (M+H).

Preparation of((2-((4-(dimethylamino)butanoyl)oxy)acetyl)azanediyl)bis(ethane-2,1-diyl)ditetra-decanoate(HEDC-M1)

Step 1: Preparation of Intermediate 1:((2-(benzyloxy)acetyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate

Synthesis of azanediylbis(ethane-2,1-diyl)ditetradecanoate TFA saltpreviously described. Azanediylbis(ethane-2,1-diyl)ditetradecanoate TFAsalt was stirred in DCM (25 mL) with 10% K₂CO₃ (12.5 mL) at 0-5° C.After 30 min, the organic layer was isolated and the aqueous layer wasfurther extracted with DCM (12 mL). The combined organic phases arestirred with MgSO₄ for 30 min at 0-5° C., filtered, washed with DCM (12mL). To the combined filtrates are added benzyloxy acetic acid (402 uL,2.81 mmol), EDC HCl salt (673 mg, 3.51 mmol), and DMAP (29 mg, 0.234mmol). The suspension was allowed to stir at room temperature overnight.Next day, H₂O (25 mL) and MeOH (12 mL) was added and after stirring for10 min the clear organic phase was isolated. The turbid aqueous phasewas extracted with DCM (25 mL). The combined organic extracts dried withMgSO₄, filtered and concentrated. Purification by silica gelchromatography eluating with a hexanes/ethyl acetate gradient yielded((2-(benzyloxy)acetyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate(1.28 g).

Step 2: Preparation of Intermediate 2:((2-hydroxyacetyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate

((2-(benzyloxy)acetyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate(1.28 g, 1.80 mmol) was dissolved in a round-bottom flask with MeOH (20mL). The flask was capped and flushed with argon. 10% Pd/C (135 mg) wasadded and the flask was once again flushed with argon. All air wasremoved via vacuum pump and then a 8″ balloon filled with H₂ gas wasadded. Reaction was allowed to stir vigorously at room temperature.After 30 min, the reaction mixture was filtered (celite), washed withmethanol, concentrated to residue, taken up in DCM (25 mL) and 10% K₂CO₃(25 mL). The mixture was stirred for 15 min and then the organic layerisolated. The aqueous wash was back extracted with DCM (15 mL). Thecombined organics were dried with MgSO₄, filtered and concentrated toyield ((2-hydroxyacetyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate(990 mg).

Step 3: Preparation of HEDC-M1:((2-((4-(dimethylamino)butanoyl)oxy)acetyl)azanediyl)bis(ethane-2,1-diyl)di-tetradecanoate

((2-hydroxyacetyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate (990mg, 1.70 mmol) was stirred in DCM (20 mL) and 4-Dimethylamino-butyricacid (268 mg), EDC HCl salt (487 mg) and DMAP (21 mg) was added. Thesuspension was allowed to stir at room temperature overnight. Next day,H₂O (20 mL) and MeOH (10 mL) added and after stirring for 10 minutes theclear organic phase was isolated. The turbid aqueous phase was extractedwith DCM (20 mL). The combined organic extracts dried with MgSO₄,filtered and concentrated. Crude material was purified by silica gelchromatography eluating with a DCM/MeOH gradient. Pooled andconcentrated fractions were concentrated and taken up with DCM (25 mL)and PBS (pH=11, 25 mL). The mixture was stirred at room temperature for15 min. Afterwards, the organic phase was isolated, and the aqueousphase was extracted again with DCM (25 mL). The combined organics aredried (MgSO₄), filtered, and concentrated to yield((2-((4-(dimethylamino)butanoyl)oxy)acetyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate(672 mg). LCMS ESI+: m/z 697.6 (M+H).

Preparation of(Z)-((5-(dimethylamino)pentanoyl)azanediyl)bis(ethane-2,1-diyl)dioleate(C104-DO)

Synthesis of (Z)-azanediylbis(ethane-2,1-diyl)dioleate TFA saltpreviously described. (Z)-azanediylbis(ethane-2,1-diyl)dioleate TFA salt(1.50 g, 2.37 mmol) was stirred in DCM (20 mL) with 10% K₂CO₃ (10 mL) at0-5° C. After 30 min, the organic phase was isolated and the aqueousphase was further extracted with DCM (10 mL). The combined organicphases are stirred with MgSO4 for a period of 30 min at 0-5° C.,filtered and washed with DCM (15 mL). To the combined filtrates areadded 5-(dimethylamino)pentanoic acid (516 mg, 2.84 mmol), EDC HCl salt(681 mg, 3.55 mmol), DMAP (29 mg, 0.237 mmol) and the suspension wasstirred overnight at room temperature, after which period of time becameclear solution was formed. Next day, H₂O (20 mL) and MeOH (10 mL) addedand after stirring for 10 min, the clear organic phase was isolated. Theturbid aqueous phase was extracted with DCM. The combined organics aredried (MgSO4), filtered and concentrated. After purification by silicagel chromatography eluating with a DCM/MeOH gradient, the pooled andconcentrated fractions are taken up with DCM (25 mL) and PBS (pH=11, 25mL). The mixture was stirred at room temperature for ˜10 min.Afterwards, the organic phase was isolated out and the aqueous phase wasextracted again with DCM (15 mL). The combined organics are dried(MgSO₄), filtered, and concentrated to yield(Z)-((5-(dimethylamino)pentanoyl)azanediyl)bis(ethane-2,1-diyl)dioleate(1.10 g). LCMS ESI+: m/z 761.7 (M+H).

Preparation of((5-((dimethylamino)methyl)thiophene-2-carbonyl)azanediyl)bis(ethane-2,1-diyl)di-tetradecanoate(T104)

Synthesis of azanediylbis(ethane-2,1-diyl)ditetradecanoate TFA saltpreviously described. Azanediylbis(ethane-2,1-diyl)ditetradecanoate TFAsalt (1004 mg, 1.57 mmol) was stirred in DCM (20 mL) with 10% K₂CO₃ (20mL) at 0-5° C. After 30 min, the organic phase was isolated and theaqueous phase was further extracted with DCM (10 mL). The combinedorganics are dried with MgSO₄ for 30 min at 0-5° C., filtered and washedwith DCM (10 mL). To the combined filtrates were added((dimethylamino)methyl)thiophene-2-carboxylic acid (350 mg, 1.89 mmol),EDC HCl salt (452 mg, 2.36 mmol), and DMAP (19.2 mg, 0.157 mmol). Thesuspension was allowed to stir at room temperature overnight. Next day,H₂O (20 mL) and MeOH (10 mL) were added and after stirring for 10 min,the clear organic phase was isolated. The turbid aqueous phase wasextracted with DCM (25 mL). The combined organics were dried (MgSO₄),filtered, and concentrated. After purification by silica gelchromatography eluating with a hexanes/ethyl acetate gradient, thepooled and concentrated fractions were taken up with DCM (20 mL) and PBS(pH=11, 20 mL). The mixture was stirred at room temperature for ˜10 min.Afterwards, the organic phase was isolated and the aqueous phase wasextracted again with DCM (15 mL). The combined organics was dried(MgSO₄) for a period 30 min, filtered, washed with DCM, and concentratedto yield((5-((dimethylamino)methyl)thiophene-2-carbonyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate(482 mg). LCMS ESI+: m/z 693.6 (M+H).

Preparation of(Z)-((((2-(dimethylamino)ethyl)thio)carbonyl)azanediyl)bis(ethane-2,1-diyl)dioleate(TU104-DO)

Synthesis of(Z)-((tert-butoxycarbonyl)azanediyl)bis(ethane-2,1-diyl)dioleatepreviously described.(Z)-((tert-butoxycarbonyl)azanediyl)bis(ethane-2,1-diyl)dioleate (4.20g, 5.72 mmol) was dissolved in DCM (20 mL) and cooled to 0° C. in an icebath. TFA (20 mL) was added and the mixture was allowed to stir under ablanket of inert gas for 20 min. Afterwards, the reaction mixture wasconcentrated in vacuo. The residue was partitioned between 10% K₂CO₃ (20mL) and DCM (20 mL), and stirred in an ice bath for 20 min. The organiclayer was isolated, dried (MgSO₄), and filtered. Diphosgene (1.38 mL,11.4 mmol) was added to (Z)-azanediylbis(ethane-2,1-diyl)dioleatematerial in DCM and stirred under a blanket of inert gas at roomtemperature. Next day, DCM and excess diphosgene were removed in vacuo.2-(dimethylamino)ethane thiol HCl salt (4.05 g, 28.6 mmol) was taken upin DCM (50 mL) and triethylamine (5.2 mL, 37.2 mmol) and added to(Z)-((chlorocarbonyl)azanediyl)bis(ethane-2,1-diyl)dioleate residue.Material was allowed to stir overnight at room temperature. Next day,diluted with DCM and washed with 0.3M HCl (75 mL), H₂O (75 mL) and 10%K₂CO₃ (75 mL). Back extracted all aqueous washes with DCM (25 mL). Thecombined organics were dried over MgSO₄, filtered and concentrated.Purification by silica gel chromatography eluating with DCM/MeOHgradient yielded(Z)-((((2-(dimethylamino)ethyl)thio)carbonyl)azanediyl)bis(ethane-2,1-diyl)dioleate(1.90 g). LCMS ESI+: m/z 765.7 (M+H).

Preparation of(((3-(dimethylamino)propoxy)carbonyl)azanediyl)bis(ethane-2,1-diyl)ditetradeca-noate(CB104)

Synthesis of azanediylbis(ethane-2,1-diyl)ditetradecanoate previouslydescribed. Diphosgene (266 uL, 2.2 mmol) was added todimethylaminopropanol (413 mg, 4.00 mmol) in DCM (10 mL) and stirredunder a blanket of inert gas at room temperature for 4 h. The DCM andexcess diphosgene was removed in vacuo andazanediylbis(ethane-2,1-diyl)ditetradecanoate was added. Theround-bottom flask was flushed with argon and DCM (10 mL) andtriethylamine (859 uL, 6.16 mmol) was added. Material was allowed tostir overnight at room temperature. The reaction mixture was thendiluted with DCM and washed with 0.3M HCl (75 mL), H₂O (75 mL) and 10%K₂CO₃ (75 mL). Back extracted all aqueous washes with DCM (25 mL). Thecombined organics were dried over MgSO₄, filtered and concentrated.Purification by silica gel chromatography eluating with DCM/MeOHgradient yielded(((3-(dimethylamino)propoxy)carbonyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate(87 mg).). LCMS ESI+: m/z 655.59 (M+H).

Preparation of(((2-(dimethylamino)ethoxy)carbonyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate(CA104)

Step 1: Preparation of Intermediate 1:(((4-nitrophenoxy)carbonyl)azanediyl)bis(ethane-2,1-diyl)ditetradeca-noate

Synthesis of azanediylbis(ethane-2,1-diyl)ditetradecanoate TFA saltpreviously described. Azanediylbis(ethane-2,1-diyl)ditetradecanoate TFAsalt was dissolved in dry DCM (10 mL) and triethylamine (654 uL, 4.69mmol) was added. The reaction vessel was flushed with inert gas and4-nitrophenyl chloroformate was added. Material was allowed to stir atRT overnight. The reaction mixture was quenched with water (50 mL) andDCM (50 mL). The organic layer was isolated, and the aqueous layer wasextracted further with DCM (2×50 mL). The combined organics were driedover MgSO₄, filtered and concentrated. Purification by silica gelchromatography eluating with hexanes/ethyl acetate gradient yielded(((4-nitrophenoxy)carbonyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate.

Step 2: Preparation of CA104:(((2-(dimethylamino)ethoxy)carbonyl)azanediyl)bis(ethane-2,1-diyl)ditetra-decanoate

To(((4-nitrophenoxy)carbonyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate,was added 2-dimethylaminoethanol (2 mL) and heated to 140° C. with acondensing column for 20 min. Afterwards, the crude material waspurified by silica gel chromatography eluating with a DCM/MeOH gradientto yield(((2-(dimethylamino)ethoxy)carbonyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate(38 mg). LCMS ESI+: m/z 641.7 (M+H).

Preparation of((2-(dimethylamino)acetyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate(INT-4)

Synthesis of(Z)-((2-(dimethylamino)acetyl)azanediyl)bis(ethane-2,1-diyl)dioleate wasprepared in similar fashion as i-Prop-DODC with the substitution ofdimethylglycine for 3-(dimethylamino)propionic acid. QTOF MS ESI+: m/z720.1 (M+H).

Preparation of((2-((2-(dimethylamino)ethyl)thio)acetyl)azanediyl)bis(ethane-2,1-diyl)ditetradeca-noate(S104)

Step 1: Preparation of intermediate 1:2-((2-(dimethylamino)ethyl)thio)acetic acid hydrochloride

Ethanol (500 mL) was degassed by three times evacuation to 60 mBar for1-2 minutes and repressurizing with nitrogen. Chloroacetic acid (36.9 g,0.391 mol) was added and a clear solution was formed after havingstirred for 5 minutes at 17-20° C. 2-(Dimethylamino)-ethanethiolhydrochloride (52.7 g, 0.372 mol) was added, and a clear solution wasformed after having stirred for 20 minutes at 25° C. Solid sodiumhydroxide (47.7 g, 1.19 mol) was added in portions over a period of 20minutes with cooling in order to keep the temperature below 35° C. —ashort period at 44° C. was observed. Almost immediate precipitation wasobserved. The reaction mixture was eventually heated and stirred at 40°C. for a period of two hours, at which point TLC indicated completion ofreaction. Celite 545 (74 g) was added, and the mixture was filteredthrough a G3 glass-sintered plate over 6 minutes, washing with ethanol(2×105 mL). The combined cloudy filtrates are evaporated from a 50° C.bath to give 110 g of white solid. The solid was dissolved in water (250mL) then the pH was adjusted from 13.1 (temp. 30° C.) to 10.5 (temp 31°C.), using concentrated HCl (5.5 mL) to give a very pale yellowsolution. The aqueous phase was washed with DCM (3×100 mL) to remove thedisulphide impurity (all 3 washes required). Concentrated HCl was addedto the aqueous phase (pH 10.7, temp. 22° C.) until the pH was 1.4 (57.5mL added, temp. 35° C.). The aqueous phase was washed with DCM (100 mL)and then concentrated to dryness (bath temperature 55° C.). Toluene (250mL) was added, the mixture was concentrated to dryness (bath temperature55° C.) and this was repeated once to give a wet white solid (98 g).Acetonitrile (750 mL) was added to the solid, the mixture was stirred at55° C. for a period of 45 minutes, and then filtered through a G3glass-sintered plate. Acetonitrile (250 mL) was added to the filtercake, the mixture was stirred at 55° C. for a period of 25 minutes andthen filtered through a G3 glass-sintered plate, washing withacetonitrile (50 mL). The combined filtrates are concentrated to 300 mL,resulting in a heavy, white precipitate. The mixture was cooled undernitrogen and stirred at 0° C. for a period of 30 minutes. Theprecipitate was isolated by filtration through a G3 glass sinteredplate, and the filter cake was washed with cold acetonitrile (100 mL).Drying under reduced pressure for 3 days gives 47.0 g (63%) of2-((2-(dimethylamino)ethyl)thio)acetic acid hydrochloride

Step 2: Preparation of S104:((2-((2-(dimethylamino)ethyl)thio)acetyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate

Synthesis of azanediylbis(ethane-2,1-diyl)ditetradecanoate TFA saltpreviously described. Azanediylbis(ethane-2,1-diyl)ditetradecanoate TFAsalt (152 g, 238 mmol) was stirred with DCM (2.3 L) and 10% potassiumbicarbonate (1.15 L) at 0-5° C. The organic phase was separated and theaqueous phase was further extracted with DCM (1.15 L). The combinedorganic phases were stirred with magnesium sulfate hydrate (236 g) for aperiod of 30 minutes at 0-5° C., filtrated and washed with DCM (1.15 L).To the combined filtrates was added2-((2-(dimethylamino)ethyl)thio)acetic acid hydrochloride (57.0 g, 285mmol), EDC hydrochloride (68.4 g, 357 mmol) and DMAP (2.91 g, 23.8mmol), and the thin suspension was stirred overnight at ambienttemperature, after which period of time a clear solution was formed.water (2.3 L) and methanol (460 mL) are added and after having stirredfor a period of 10 minutes the clear organic phase was separated. Theturbid aqueous phase (pH 3.0) was extracted with DCM (575 mL). Thecombined organic extracts were concentrated yielding 143 g of crudematerial as the hydrochloride salt. The crude material (142.6 g) wastransferred to a distillation flask with DCM (500 mL), and ethyl acetate(1 L) was added. The solution was heated to distillation at atmosphericpressure, and distillation was continued over a period of 70 minutes inorder to obtain a temperature of the residue of 76° C. A total volume of1.4 L was obtained by addition of ethyl acetate (800 mL), and ethanol(70 mL) was added. The clear solution at 50° C. was cooled to 37° C. andseed crystals were added. Having observed initiation of significantcrystallization over a period of 10 minutes at 37-35° C., the suspensionwas cooled and stirred at 0° C. overnight and the precipitate wasisolated by filtration, and washed with cold ethyl acetate (210 mL).Drying to a constant weight at ambient temperature in oil pump vacuumover a period of 4.5 hours gave 134 g of recrystallized material as thehydrochloride salt, white crystalline solid.

Tripotassium phosphate (85 g, 0.40 mol) and dipotassium hydrogenphosphate (226 g, 1.30 mol) were added to purified water (1.7 L), andthe solution formed with pH 10.9 was cooled to 18-20° C. DCM (1.3 L) andrecrystallized S104 hydrochloride (133.0 g, 0.188 mol) are added, andthe mixture was stirred for a period of 10 min minutes. A clear organicphase was separated at moderate rate (over a period of 35 minutes), andthe turbid aqueous phase was further extracted with DCM (650 mL). Thecombined organic phases were stirred with magnesium sulfate hydrate (65g) for a period of 40 minutes, and the mixture was filtered, washingwith DCM (200 mL). The combined filtrates were evaporated from a 50° C.water bath under reduced pressure (down to 20 mBar, at which pressureevaporation was continued for one hour). Additional evaporation from a15-20° C. water bath at oil pump vacuum, resulted in 126 g partiallysolidified oil. Cooling in −20° C. cooling bath gave completesolidification, and after drying at −20° C. under vacuum for two days weobtained 126 g of((2-((2-(dimethylamino)ethyl)thio)acetyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate,also known as S104. HPLC indicates 98.1% purity. QTOF MS ESI+: m/z 671.6(M+H).

Preparation of(9Z,9′Z)-((2-((2-(dimethylamino)ethyl)thio)acetyl)azanediyl)bis(ethane-2,1-diyl)bis(tetradec-9-enoate)(S104-DMO)

Step 1: Preparation of Intermediate 1:(9Z,9′Z)-((tert-butoxycarbonyl)azanediyl)bis(ethane-2,1-diyl)bis(tetradec-9-enoate)

N-Boc-diethanolamine (454 mg, 2.21 mmol), myristoleic acid (1000 mg,4.42 mmol) and DMAP (54 mg, 0.442 mmol) was dissolved in DCM (25 mL) andplaced in a water bath at ambient temperature in a round-bottom flaskwas flushed with inert gas. EDC HCl salt (932 mg, 4.86 mmol) was addedin 3 portions over 5 min. The reaction was allowed to stir overnight atroom temperature under a blanket of inert gas. Next day, added H₂O (25mL) and stirred for 10 min. The organic layer was isolated and theaqueous layer was further extracted with DCM (50 mL). The combinedorganics were dried (MgSO₄) for 10 min, filtered and concentrated.Purification by silica gel chromatography eluating with a hexane/ethylacetate gradient yielded(9Z,9′Z)-((tert-butoxycarbonyl)azanediyl)bis(ethane-2,1-diyl)bis(tetradec-9-enoate)(1.10 g).

Step 2: Preparation of Intermediate 2:(9Z,9′Z)-azanediylbis(ethane-2,1-diyl)bis(tetradec-9-enoate)

To(9Z,9′Z)-((tert-butoxycarbonyl)azanediyl)bis(ethane-2,1-diyl)bis(tetradec-9-enoate)(1100 mg, 1.77 mmol) in a round-bottom flask was added DCM (10 mL) andplaced into an ice-bath. TFA (10 mL) was added and the mixture wasallowed to stir for 20 min. The reaction mixture was then concentrated.Toluene was added to the residue to aid in azeotroping off excess TFA.The residue was placed back in the ice bath and PBS (pH=11, 25 mL) andDCM (25 mL) were added. The mixture was stirred for 15 min and theorganic layer was then isolated. The turbid aqueous layer was extractedwith DCM (10 mL). The combined organics were dried (MgSO4) at 0° C. for15 min, filtered and concentrated to yield(9Z,9′Z)-azanediylbis(ethane-2,1-diyl)bis(tetradec-9-enoate) (923 mg).

Step 3: Preparation of S104-DMO:(9Z,9′Z)-((2-((2-(dimethylamino)ethyl)thio)acetyl)azanediyl)bis(ethane-2,1-diyl)bis(tetradec-9-enoate)

A mixture of(9Z,9′Z)-azanediylbis(ethane-2,1-diyl)bis(tetradec-9-enoate) (923 mg,1.77 mmol), 2-((2-(dimethylamino)ethyl)thio)acetic acid (346 mg, 2.12mmol) and EDC HCl salt (509 mg, 2.66 mmol) suspended in DCM (10 mL).DMAP (21.6 mg, 0.177 mmol) was added and the mixture was allowed to stirat room temperature overnight. Next day, H₂O (10 mL) and MeOH (10 mL)was added and after stirring for 10 min, the clear organic phase wasisolated. The turbid aqueous phase was extracted with DCM (2×20 mL). Thecombined organic extracts are dried with MgSO₄, filtered andconcentrated. After purification by silica gel chromatography eluatingwith a DCM/MeOH, the pooled and concentrated fractions were taken up inDCM (25 mL) and PBS (pH=11, 25 mL). The mixture was stirred at roomtemperature for ˜10 min. Afterwards, the organic phase was isolated andthe aqueous phase was extracted again with DCM (2×15 mL). The combinedorganics phase were dried (MgSO₄), filtered, and concentrated to yield(9Z,9′Z)-((2-((2-(dimethylamino)ethyl)thio)acetyl)azanediyl)bis(ethane-2,1-diyl)bis(tetradec-9-enoate)(589 mg). LCMS ESI+: m/z 667.6 (M+H).

Preparation of(R)-((1-methylpyrrolidine-2-carbonyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate(Pro-DC)

Step 1: Preparation of Pro-DC:(R)-((1-methylpyrrolidine-2-carbonyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate

Synthesis of azanediylbis(ethane-2,1-diyl)ditetradecanoate TFA saltpreviously described. Azanediylbis(ethane-2,1-diyl)ditetradecanoate TFAsalt (1000 mg, 1.56 mmol) was stirred with DCM (10 mL) andN-Methyl-L-Proline (228 mg, 1.77 mmol), HOBt H₂O (239 mg, 1.77 mmol) wasadded. NMM (365 uL, 3.32 mmol) was added and the solution became mostlyclear. A suspension of EDC hydrochloride (518 mg, 2.70 mmol), NMM (257uL, 2.34 mmol) and DMAP (19 mg, 0.156 mmol) in DCM (10 mL) was added andthe mixture was stirred for about 12 hours at ambient temperature, afterwhich period of time a clear solution was formed. Thereafter, themixture was diluted with DCM (50 mL) and washed with 10% K₂CO₃, aqueous(60 mL). The organics were dried with MgSO₄, filtered and concentrated.The resulting compound was purified crude by Silica Gel chromatography,eluting with a (0-10) % methanol in DCM gradient to yield(R)-((1-methylpyrrolidine-2-carbonyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate.LCMS ESI+: m/z 637.6 (M+H).

Preparation of(9Z,9′Z,12Z,12′Z)-((2-((2-(dimethylamino)ethyl)thio)acetyl)azanediyl)bis(ethane-2,1-diyl)bis(octadeca-9,12-dienoate)(S104-DLin)

Step 1: Preparation of Intermediate 1:(9Z,9′Z,12Z,12′Z)-((tert-butoxycarbonyl)azanediyl)bis(ethane-2,1-diyl)bis(octadeca-9,12-dienoate)

N-Boc-diethanolamine (5 g, 24.4 mmol), linoleic acid (14.4 g, 51.2 mmol)and was dissolved in DCM (100 mL). EDC HCl salt (10.3 g, 53.7 mmol) wasadded followed by DMAP (596 mg, 4.88 mmol). The reaction was allowed tostir for about 12 hours at room temperature under a blanket of inertgas. Thereafter, 50 mL of water and 50 mL of methanol were added, andthe mixture was stirred for 10 min. The organic layer was isolated andthe aqueous layer was further extracted with DCM (150 mL). The combinedorganics were dried (MgSO₄) for 10 min, filtered and concentrated.Purification was by silica gel chromatography, eluating with ahexane/ethyl acetate gradient yielded(9Z,9′Z,12Z,12′Z)-((tert-butoxycarbonyl)azanediyl)bis(ethane-2,1-diyl)bis(octadeca-9,12-dienoate)(15.9 g).

Step 2: Preparation of Intermediate 2:(9Z,9′Z,12Z,12′Z)-azanediylbis(ethane-2,1-diyl)bis(octadeca-9,12-dienoate)

To(9Z,9′Z,12Z,12′Z)-((tert-butoxycarbonyl)azanediyl)bis(ethane-2,1-diyl)bis(octadeca-9,12-dienoate)(5.33 g, 7.30 mmol) in a round-bottom flask was added DCM (50 mL) andplaced into an ice-bath. TFA (50 mL) was added and the mixture wasallowed to stir for 30 min. The reaction mixture was then concentrated.Toluene was added to the residue to aid in azeotroping off excess TFA.The residue was placed back in the ice bath and 10% K₂CO₃ (50 mL) andDCM (50 mL) were added. The mixture was stirred for 15 min and theorganic layer isolated. The turbid aqueous layer was extracted with DCM(20 mL). The combined organics were dried (MgSO4), filtered andconcentrated to yield(9Z,9′Z,12Z,12′Z)-azanediylbis(ethane-2,1-diyl)bis(octadeca-9,12-dienoate)(Quantitative).

Step 3: Preparation of S104-DLin:(9Z,9′Z,12Z,12′Z)-((2-((2-(dimethylamino)ethyl)thio)acetyl)azanediyl)bis(ethane-2,1-diyl)bis(octadeca-9,12-dienoate)

A mixture of(9Z,9′Z,12Z,12′Z)-azanediylbis(ethane-2,1-diyl)bis(octadeca-9,12-dienoate)(4.68 g, 7.30 mmol), 2-((2-(dimethylamino)ethyl)thio)acetic acid (1.43g, 8.76 mmol) and EDC HCl salt (2.10 g, 10.95 mmol) were suspended inDCM (100 mL). DMAP (89 mg, 0.73 mmol) was added, stirred at roomtemperature for 12 hours. Fifty mL each of water and methanol were addedand, after stirring for 10 minutes, the clear organic phase wasisolated. The turbid aqueous phase was extracted with DCM (2×20 mL) andwashed combined organic extracts with PBS (pH=11, 100 mL). The productwas dried with MgSO₄, filtered and concentrated. Purification was bysilica gel chromatography eluating with a MeOH in DCM gradient. Pooledand concentrated fractions yielded(9Z,9′Z,12Z,12′Z)-((2-((2-(dimethylamino)ethyl)thio)acetyl)azanediyl)bis(ethane-2,1-diyl)bis(octadeca-9,12-dienoate)(4.3 g). LCMS ESI+: m/z 775.9 (M+H).

Preparation of(9Z,9′Z,12Z,12′Z)-((2-((2-(dimethylamino)ethyl)thio)acetyl)azanediyl)bis(ethane-2,1-diyl)bis(octadeca-9,12-dienoate)(TU104-DLin)

Step 1: Preparation of TU104-Dlin:(9Z,9′Z,12Z,12′Z)-((2-((2-(dimethylamino)ethyl)thio)acetyl)azanediyl)bis(ethane-2,1-diyl)bis(octadeca-9,12-dienoate)

Synthesis of(9Z,9′Z,12Z,12′Z)-azanediylbis(ethane-2,1-diyl)bis(octadeca-9,12-dienoate)previously described. Trichloromethyl chloroformate (aka diphosgene)(740 uL,) was added to a solution(9Z,9′Z,12Z,12′Z)-azanediylbis(ethane-2,1-diyl)bis(octadeca-9,12-dienoate)(2.6 g) in dry DCM (40 mL) and stirred under a blanket of argon at roomtemperature for 12 hours. DCM and excess diphosgene were removed invacuo. 2-(dimethylamino)ethane thiol HCl salt (2.9 g), DCM (40 mL) andtriethylamine (3.7 mL) were then added. After 16 h at room temperaturethe reaction mixture was diluted with DCM and washed with 10% K₂CO₃ (75mL), dried (MgSO₄), filtered, and concentrated. Purification waspurified by silica gel chromatography eluating with ethyl acetatefollowed by a DCM/MeOH gradient to yield(9Z,9′Z,12Z,12′Z)-((2-((2-(dimethylamino)ethyl)thio)acetyl)azanediyl)bis(ethane-2,1-diyl)bis(octadeca-9,12-dienoate)(850 mg). LCMS ESI+: m/z 761.9 (M+H).

Formation of Non-diVA siRNA Containing Liposomes.

Ionizable lipid, DOPE, cholesterol, and a PEG-conjugated lipid weresolubilized in absolute EtOH (200 proof) at a final weight concentrationof ˜4.4 mg/mL. The siRNA was solubilized in citrate buffer at aconcentration ˜0.26 mg/mL and the temperature was adjusted to 35-40° C.The ethanol/lipid mixture was then added to the siRNA-containing bufferwhile stirring to spontaneously form siRNA loaded liposomes. Lipids werecombined with siRNA to reach a final total lipid to siRNA ratio of 7:1to 14:1 (wt:wt). The siRNA loaded liposomes were diafiltered against 10×volumes of PBS to remove ethanol and exchange the buffer. Final productwas filtered through 0.22 μm, sterilizing grade, filter for bioburdenreduction. This process yielded liposomes with a mean particle diameterof 40-100 nm, PDI <0.2, and >85% RNA encapsulation (entrapment)efficiency.

Formation of siRNA containing liposomes co-solubilized with diVA.

siRNA-diVA-Liposome formulations were prepared using the methoddescribed above. diVA-PEG-diVA was co-solubilized in absolute ethanolwith the other lipids (ionizable lipid, DOPE, cholesterol, andPEG-conjugated lipids) prior to addition to the siRNA containing buffer.Molar content of diVA-PEG-diVA ranged from 0.1 to 5 mol %. This processyielded liposomes with a mean particle diameter of 40-100 nm, PDI <0.2,and >85% entrapment efficiency.

Formation of siRNA Containing Liposomes with Ionizable and CationicLipids.

siRNA-diVA-Liposome formulations and siRNA-Liposome formulations wereprepared using the method described above. Cationic lipid wasco-solubilized in absolute ethanol with the other lipids (ionizablelipid, DOPE, cholesterol, PEG-conjugated lipids, and diVA-PEG-diVA)prior to addition to the siRNA containing buffer. Molar content ofcationic lipid ranged from 5 to 40 mol %. This process yielded liposomeswith a mean particle diameter of 40-100 nm, PDI <0.2, and >85%entrapment efficiency.

In Vitro (pHSC, gp46 KD @ 20 nM) Efficacy

PHSCs in 96-well plate were incubated with formulation that composed ofeither ionizable lipid formulation C104, ionizable lipid formulationTu104, or combination formulations with different ratio of ionizablelipids (C104:Tu104). After 30 minutes, medium was replaced with freshgrowth medium. Forty eight hours later, cells were lysed and gp46 andGAPDH mRNA levels measured by quantitative RT-PCR (TaqMan®) assay, andgp46 levels were normalized to GAPDH levels. Normalized gp46 levels wereexpressed as percent of mock control cells. Error bars indicate standarddeviations (n=3). Results are depicted in FIG. 1. As the resultsdemonstrate, the combination of two ionizable lipids of the descriptionresulted in observed synergistic reduction in gene expression.

Similar experiments were carried out with other ionizablelipid:ionizable lipid and ionizable lipid:cationic lipid combinations.Results for S104-DO:Tu104-DO combinations are depicted in FIG. 2.Results for HEDODC:Tu104 combinations are depicted in FIG. 3.Combinations of both ionizable lipids and ionizable:cationic lipidsagain resulted in synergistic reduction in gene expression.

In Vivo (DMNQ) Efficacy:

In vivo activity of target formulation was evaluated in the short-termliver damage model (referred to as the Quick Model or DMNQ). In thismodel, the short-term liver damage induced by treatment with ahepatotoxic agent such as dimethylnitrosamine (DMN) is accompanied bythe elevation of gp46 mRNA levels. To induce these changes, maleSprague-Dawley rats were injected intraperitoneally with DMN on sixconsecutive days. At the end of the DMN treatment period, the animalswere randomized to groups based upon individual animal body weight.Formulations was administered as a single IV dose, one hour after thelast injection of DMN. Twenty four hours after, liver lobes were excisedand both gp46 and MRPL19 mRNA levels were determined by quantitativeRT-PCR (TaqMan) assay. mRNA levels for gp46 were normalized to MRPL19levels. Results are depicted in FIG. 4. Several combinations ofionizable:cationic lipids achieved 50% reduction in gene expression fora single, 0.5 mg per kg of animal body weight, dose of encapsulatedsiRNA.

In Vitro Toxicology Data, In Vitro Cytotoxicity (HepG2 @ 200 nM)

Addition of 20 mol % of S 104 into formulations of the descriptionimproved cell viability in a HepG2 cytotoxicity assay improved from 27%to 52%.

Formulation % viability 40:30:25:5:2(HEDC:DOPE:Cholesterol:Peg-lipid:DiVA) 27 ± 5.3% 20:20:30:25:5:2(HEDC:S104:DOPE:Cholesterol:Peg- 52 ± 10%  lipid:DiVA)

HepG2 Cytotoxicity Assay Description:

HepG2 cells, an adherent cell line derived from human hepatocellularcarcinoma, was cultured in MEM/EBSS (Hyclone, Logan, Utah,Cat#SH30024.01) supplemented with 10% FBS (Hyclone, Logan, UtahCat#SH30910). HepG2 cells were seeded in 96-well Optilux black plates(BD Falcon, Cat #BD353220) at 5000 cells/well overnight. Formulationswere added to each well to final indicated siRNA concentration (n=3). At48 h post formulation addition, cell viability was determined usingCellTiter-Glo Luminescent Cell Viability Assay Kit (Promega, Cat #G7572)following manufacture's instruction. Chemiluminescent signal weremeasured on Clarity Luminescence Microplate Reader (502-Biotek,Winooski, Vt.). Viability was calculated based on % of chemiluminescentsignal in formulation treated well normalized against mock treatedwells.

In Vivo Toxicology Data

The HEDC:S104 (20:20) formulation of the present description isexceptionally well tolerated as shown in toxicity studies. No toxicitywas observed when the formulation was injected intravenously in rats andmonkey at doses up to 25 mg/kg and 12 mg/kg, respectively, which isconsidered by those skilled in the art to be superior.

Transfection with Formulations of the Description:

Transfection method is same for LX-2 and pHSCs. The liposomeformulations or lipoplex formulations of the description were mixed withgrowth medium at desired concentrations. 100 μl of the mixture was addedto the cells in 96-well plate and cells were incubated for 30 min at 37°C. in the incubator with 5% CO₂. After 30 min, medium was replaced withfresh growth medium. After 48 h of transfection, cells were processedusing Cell-to-Ct lysis reagents (Applied Biosystems) according to themanufacturer's instructions.

Quantitatve (q) RT-PCR for Measuring HSP47 mRNA Expression

HSP47 and GAPDH TaqMan° ® assays and One-Step RT-PCR master mix werepurchased from Applied Biosystems. Each PCR reaction contained thefollowing composition: One-step RT-PCR mix 5 μl, TaqMan® RT enzyme mix0.25 μl, TaqMan® gene expression assay probe (HSP47) 0.25 μl, TaqMan®gene expression assay probe (GAPDH) 0.5 μl, RNase free water 3.25 μl,Cell lysate 0.75 μl, Total volume of 10 μl. GAPDH was used as endogenouscontrol for the relative quantification of HSP47 mRNA levels.Quantitative RT-PCR was performed in ViiA 7 realtime PCR system (AppliedBiosciences). All values were normalized to the average HSP47 expressionof the mock transfected cells and expressed as percentage of HSP47expression compared to mock.

What is claimed:
 1. A ionizable lipid compound of formula I:

wherein n and m are independently 1, 2, 3, or 4; R₁ and R₂ areindependently C₁₀₋₁₈alkyl or C₁₂₋₁₈alkenyl; X is —CH₂—, S, O, N, orabsent; L is C₁₋₄alkylene, —O—C(O)—C₁₋₄alkylene, —S(O)₂—C₁₋₄alkylene,

or a pharmaceutically acceptable salt form thereof.
 2. The compound ofclaim 1, wherein n and m are 1 or
 2. 3. The compound of claim 1, whereinX is absent.
 4. The compound of claim 3, wherein L is C₁₋₄alkylene. 5.The compound of claim 4, wherein L is selected from the group consistingof —CH₂—, —CH₂—CH₂—, —CH₂—CH₂CH₂—, —CH₂—CH₂CH₂CH₂—,


6. The compound of claim 1, wherein X is —CH₂—.
 7. The compound of claim6, wherein L is —S—C₁₋₄alkylene.
 8. The compound of claim 7, wherein Lis —S—CH₂— or —S—CH₂—CH₂—.
 9. The compound of claim 6, wherein L is—S(O)₂—C₁₋₄alkylene.
 10. The compound of claim 9, wherein L is—S(O)₂—CH₂—CH₂—.
 11. The compound of claim 6, wherein L is—O—C₁₋₄alkylene.
 12. The compound of claim 11, wherein L is —O—CH₂—CH₂—or —O—CH₂—CH₂—CH₂—.
 13. The compound of claim 6, wherein L is—O—C(O)—C₁₋₄alkylene.
 14. The compound of claim 13, wherein L is—O—C(O)—CH₂—CH₂—CH₂—.
 15. The compound of claim 1, wherein X is S or O.16. The compound of claim 15, wherein L is C₁₋₄alkylene.
 17. Thecompound of claim 16, wherein L is —CH₂—CH₂—.
 18. The compound of claim1, wherein R₁ and R₂ are each C₁₀₋₁₈alkyl.
 19. The compound of claim 18,wherein R₁ and R₂ are each C₁₃alkyl.
 20. The compound of claim 1,wherein R₁ and R₂ are each C₁₂₋₁₈alkenyl.
 21. The compound of claim 20,wherein R₁ and R₂ are each C₁₃₋₁₇alkenyl.
 22. The compound of claim 21,wherein R₁ and R₂ are each oleyl or linoleyl.
 23. A compositioncomprising a cationic lipid and an ionizable cationic lipid in a lipidnanoparticle comprising a bilayer of lipid molecules.
 24. A compositioncomprising a compound of claim 1 in a lipid nanoparticle comprising abilayer of lipid molecules.
 25. The composition of claim 24, wherein thecompound is 5 to 50 mol % of the lipid molecules.
 26. The composition ofclaim 24, further comprising a cationic lipid.
 27. The composition ofclaim 26, wherein the cationic lipid is


28. The composition of claim 26, wherein the cationic lipid is 5 to 40mol % of the lipid molecules.
 29. The composition of claim 26, whereinthe molar ratio of the ionizable lipid and the cationic lipid is about5:35 to about 35:5.